Methods for protecting allogeneic islet transplant using soluble CTLA4 mutant molecules

ABSTRACT

The present invention is a method of inhibiting islet cell transplant rejection particular, to treat diabetes, such as type-1 and type-2 diabetes, by administering to a subject an effective amount of a soluble CTLA4 mutant molecule. One example of soluble CTLA4 mutant molecule is L104EA29YIg.

This application claims priority to provisional application, U.S. Ser.No. 60/293,402, filed May 23, 2001, the contents of which are herebyincorporated by reference in their entirety into this application.

Throughout this application various publications are referenced. Thedisclosures of these publications are hereby incorporated by referencein their entirety into this application in order to more fully describethe state of the art to which the invention pertains.

FIELD OF THE INVENTION

The present invention relates generally to the field of inhibiting isletcell transplant rejection. In particular, the invention relates tomethods for treating diabetes, including type-1 and type-2 diabetes, byadministering to a subject an effective amount of soluble CTLA4 mutantmolecules.

BACKGROUND OF THE INVENTION

Organ transplantation has emerged as a preferred method of treatment formany forms of life-threatening diseases that involve organ damage.Improved results in clinical transplantation have been achievedprimarily through the development of increasingly potent non-specificimmunosuppressive drugs to inhibit rejection responses (Lancet,345:1321-1325 (1995)). While short-term results have improved, long-termoutcomes remain inadequate. Currently, life-long immunosuppressiveagents are required to combat chronic rejection of the transplantedorgan, and the use of these agents dramatically increases the risks ofcardiovascular disease, infections and malignancies.

The development of strategies to promote the acceptance of allogeneictissues without the need for chronic immunosuppression may reduce therisk of these life-threatening complications, and greatly expand theapplication of organ, tissue and cellular transplantation for diseasessuch as the hemoglobinopathies, genetic immunodeficiencies, andautoimmune diseases.

Insulin-dependent diabetes mellitus (IDDM) is one of the most commonlyoccurring metabolic disorders in the world. In the United States, IDDMaffects approximately one in 300 to 400 people, and epidemiologicalstudies suggest that the incidence of IDDM is continuing to increase.IDDM is caused by an autoimmune response that results in the Tlymphocyte-mediated destruction of the insulin-producing islet cells ofthe pancreas.

Once the clinical symptoms of IDDM become evident, the most commonlyemployed therapy for controlling the clinical symptoms of IDDM isexogenous insulin replacement. Although insulin replacement therapyallows most IDDM patients to lead somewhat normal lives, it does notcompletely restore metabolic homeostasis, and as a result; severecomplications including dysfunctions of the eye, kidney, heart, andother organs are common in diabetic patients undergoing insulinreplacement therapy.

A long-sought treatment for IDDM patients is islet transplantation.However, transplanted insulin-producing islet cells are often rapidlydestroyed by the same autoimmune response that previously destroyed thepatients own islet cells. Of the 260 allografts transplanted since 1990,only 12.4% have resulted in insulin independence for periods of morethan one week, and only 8.25% have been insulin independent for periodsof more than one year (Linsley et al. Diabetes (1997) 46: 1120-3). Inthe majority of these procedures, the base regimen of immunosuppressionconsisted of antibody induction with an anti-lymphocyte globulincombined with cyclosporin, azathiprine, and glucocorticoids.

For any type of transplantation procedure, a balance between efficacyand toxicity is a key factor for its clinical acceptance. With respectto islet transplantation, a further concern is that many of the currentimmunosuppressive agents with particular glucocortecoids or acalcineurin inhibitor, such as Tarcolimus, damage beta cells or induceperipheral insulin resistance (Zeng et al. Surgery (1993) 113: 98-102).

A steroid-free immunosuppressive protocol (“Edmonton protocol”) thatincludes sirolimus, low dose Tarcolimus, and a monoclonal antibody (mAb)against IL-2 receptor has been used in a trial of islet transplantationalone for patients with type-1 diabetes (Shapiro, A. M. J. et al,(2000), N. Eng. J. Med., 343: 230-238).

The recent success using the “Edmonton protocol” has renewed enthusiasmfor the use of islet transplantation to treat diabetes. However,concerns regarding toxicity of the Tarcolimus may limit the applicationof this therapy in humans. Biological agents that block key T cellcostimulatory signals, in particular the CD28 pathway, are potentialalternatives to protect allogeneic islets. Examples of agents that blockthe CD28 pathway include but are not limited to soluble CTLA4 includingmutant CTLA4 molecules.

SUMMARY OF INVENTION

The present invention provides methods for treating immune systemdiseases, by administering to a subject soluble CTLA4 mutant molecules,which bind to CD80 and/or CD86 molecules on CD80 and/or CD86-positivecells, thereby inhibiting endogenous CD80 and/or CD86 molecules frombinding CTLA4 and/or CD28 on T cells, and thus, blocking key T cellcostimulatory signals, in particular the CD28 pathway.

Soluble CTLA4 mutant molecules include, but are not limited to,L104EA29Y, a molecule having mutations in the extracellular domain ofCTLA4 at alanine at position +29 and/or at leucine at position +104,wherein alanine at position 29 is substituted with tyrosine, and leucineat position 104 is substituted with glutamic acid. The CTLA4 mutantmolecules further comprise a moiety, such as an immunoglobulin molecule,that renders the mutant protein soluble.

In a preferred embodiment, the L104EA29Y is L104EA29YIg (FIG. 3).

The present invention further provides methods for inhibiting islet celltransplant rejection in a subject by administering L104EA29Y (e.g.,L104EA29YIg) to the subject undergoing islet cell transplant.

The invention also provides methods for treating diabetes in a subject,by administering an immunosuppressive regimen comprising L104EA29Y(e.g., L104EA29YIg) to the subject diagnosed with diabetes andtransplanting islet cells.

The present invention further provides pharmaceutical compositions fortreating diabetes, the compositions comprising a pharmaceuticallyacceptable carrier and soluble CTLA4 mutant, e.g., L104EA29Y.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the complete nucleotide (SEQ ID NO.: 1) and amino acidsequence (SEQ ID NO.: 2) for human CTLA4 receptor fused to theoncostatin M signal peptide. The oncostatin M signal peptide isindicated at position −25 to −1.

FIG. 2 depicts a nucleotide (SEQ ID NO.: 3) and amino acid sequence (SEQID NO.: 4) of a CTLA4Ig having a signal peptide; a wild type amino acidsequence of the extracellular domain of CTLA4 starting at methionine atposition +1 to aspartic acid at position +124, or starting at alanine atposition −1 to aspartic acid at position +124; and an Ig region.

FIG. 3 depicts a nucleotide (SEQ ID NO.: 5) and amino acid sequence (SEQID NO.: 6) of a CTLA4 mutant molecule (L104EA29YIg) comprising a signalpeptide; a mutated extracellular domain of CTLA4 starting at methionineat position +1 and ending at aspartic acid at position +124, or startingat alanine at position −1 and ending at aspartic acid at position +124;and an Ig region as described in Example 1, infra.

FIG. 4 is a line graph illustrating fasting plasma glucose level in anormal subject, as described in Example 3, infra.

FIG. 5 is a line graph illustrating fasting plasma glucose level inpancreatectomized subjects with transplanted pancreatic islet cells asdescribed in Example 3. The animals were transplanted with islet cellson day 0, and either treated with an immunosuppressive regimencontaining L104EA29YIg, and a base immunosuppressive regimen (treated),or only a base immunosuppressive regimen (control). The baseimmuosuppressive regimen contained rapamycin and anti-human IL2R.

FIG. 6 is a line graph illustrating insulin requirement in subjects withtransplanted islet cells as described in Example 3. The animals weretransplanted with islet cells on day 0, and were treated with animmunosuppressive regimen containing L104EA29YIg and a baseimmunosuppressive regimen (treated), or only a base immunosuppressiveregimen (control).

FIG. 7 is a line graph illustrating blood glucose level in anintravenous glucose tolerance test pre- and post-islet transplant, asdescribed in Example 3.

FIG. 8 depicts a schematic diagram of a vector, piLN-L104EA29Y, havingthe L104EA29YIg insert.

FIGS. 9A & 9B illustrate data from FACS assays showing binding ofL104EA29YIg, L104EIg, and CTLA4Ig to human CD80- or CD86-transfected CHOcells as described in Example 2, infra.

FIGS. 10A & 10B depicts inhibition of proliferation of CD80-positive andCD86-positive CHO cells as described in Example 2, infra.

FIGS. 11A & 11B shows that L104EA29YIg is more effective than CTLA4Ig atinhibiting proliferation of primary and secondary allostimulated T cellsas described in Example 2, infra.

FIGS. 12A-C illustrate that L104EA29YIg is more effective than CTLA4Igat inhibiting IL-2 (FIG. 12A), IL-4 (FIG. 12B), and γ-interferon (FIG.12C) cytokine production of allostimulated human T cells as described inExample 2, infra.

FIG. 13 demonstrates that L104EA29YIg is more effective than CTLA4Ig atinhibiting proliferation of phytohemaglutinin-(PHA) stimulated monkey Tcells as described in Example 2, infra.

FIGS. 14A-C are an SDS gel (FIG. 14A) for CTLA4Ig (lane 1), L104EIg(lane 2), and L104EA29YIg (lane 3A); and size exclusion chromatographsof CTLA4Ig (FIG. 14B) and L104EA29YIg (FIG. 14C).

FIGS. 15A and 15B illustrate a ribbon diagram of the CTLA4 extracellularIg V-like fold generated from the solution structure determined by NMRspectroscopy. FIG. 15B shows an expanded view of the S25-R33 region andthe MYPPPY region indicating the location and side-chain orientation ofthe avidity enhancing mutations, L104 and A29.

FIG. 16 depicts fasting blood glucose for LEA29YIg treated (A) andcontrol (B) recipients of allogeneic islets (representative animals)before and after transplantation. All animals underwent surgicalpancreatectomy at least 2 weeks before transplantation (meanpretransplant insulin requirement of 8.76±0.18 units/day) (C) Afterintraportal infusion of allogeneic islets, recipients quickly becameeuglycemic requiring no exogenous insulin posttransplant. (D) Diabetesinduction and posttransplant islet function was confirmed by intravenousglucose tolerance test before transplantation and at 1 month and 3months posttransplant, as described in Example 3, infra.

FIG. 17 depicts (A) immunohistology of functional transplanted isletconfirmed by positive staining for insulin. (B) islet from an animalreceiving control regimen surrounded by mononuclear infiltrate,indicating rejection, as described in Example 3, infra.

FIG. 18 depicts suppression of anti-donor T- and B-cell responses byL104EA29Y regimen. (A) Anti-donor IFN-γ-ELISpot response corresponds totiming of rejection in the controls (˜1 week posttransplant). (B)L104EA29Y regimen effectively suppresses the generation of anti-donor Tcell response. (C) animals receiving rapamycin anti-IL-2R mAb quicklyproduce detectable anti-donor antibody, as measured by flow cytometricmethods at the time of rejection. (D) islet recipients receiving theL104EA29Y-containing regimen fail to generate a detectable anti-donorantibody response while treated, as described in Example 3, infra.

FIG. 19 shows the nucleotide and amino acid sequences of L104EIg (SEQ IDNOs.: 7-8), as described in Example 2, infra.

FIG. 20 shows the nucleotide and amino acid sequence of L104EA29LIg (SEQID NOs.: 9-10).

FIG. 21 shows the nucleotide and amino acid sequences of L104EA29TIg(SEQ ID NOs.: 11-12).

FIG. 22 shows the nucleotide and amino acid sequences of L104EA29WIg(SEQ ID NOs.: 13-14).

FIG. 23 shows the nucleotide sequence of a CTLA4Ig (SEQ ID NO.: 15)having a signal peptide; a wild type amino acid sequence of theextracellular domain of CTLA4 starting at methionine at position +1 toaspartic acid at position +124, or starting at alanine at position −1 toaspartic acid at position +124; and an Ig region.

FIG. 24 shows the amino acid sequence of a CTLA4 μg (SEQ ID NO.: 16)having a signal peptide; a wild type amino acid sequence of theextracellular domain of CTLA4 starting at methionine at position +1 toaspartic acid at position +124, or starting at alanine at position −1 toaspartic acid at position +124; and an Ig region.

DETAILED DESCRIPTION OF THE INVENTION Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein “wild type CTLA4” has the amino acid sequence ofnaturally occurring, full length CTLA4 (U.S. Pat. Nos. 5,434,131,5,844,095, 5,851,795), or any portion thereof which binds a B7 molecule(CD80 and/or CD86), or interferes with a B7 molecule (e.g., CD80 and/orCD86) so that it blocks their binding to their ligand, or blocks theirbinding to the extracellular domain of CTLA4 or portions thereof. Inparticular embodiments, wild type CTLA4 begins with methionine atposition +1 and ends at aspartic acid at position +124, or wild typeCTLA4 begins with alanine at position −1 and ends at aspartic acid atposition +124. In other embodiments, wild type CTLA4 consists of the 187amino acids of the CTLA4 receptor as disclosed in FIG. 3 of U.S. Pat.Nos. 5,434,131, 5,844,095, 5,851,795, and shown here as FIG. 1. Wildtype CTLA4 is a cell surface protein, having an N-terminal extracellulardomain, a transmembrane domain, and a C-terminal cytoplasmic domain. Theextracellular domain binds to target antigens, such as CD80 and CD86. Ina cell, the naturally occurring, wild type CTLA4 protein is translatedas an immature polypeptide, which includes a signal peptide at theN-terminal end. The immature polypeptide undergoes post-translationalprocessing, which includes cleavage and removal of the signal peptide togenerate a CTLA4 cleavage product having a newly generated N-terminalend that differs from the N-terminal end in the immature form. Oneskilled in the art will appreciate that additional post-translationalprocessing may occur, which removes one or more of the amino acids fromthe newly generated N-terminal end of the CTLA4 cleavage product. Themature form of the CTLA4 molecule includes the extracellular domain ofCTLA4, or any portion thereof, which binds to CD80 and/or CD86.

As used herein “the extracellular domain of CTLA4” is the portion of theCTLA4 receptor that extends outside the cell membrane, and includes anyportion of CTLA4 that extends outside the cell membrane that recognizesand binds CTLA4 ligands, such as a B7 molecule (e.g., CD80 and/or CD86molecules). For example, an extracellular domain of CTLA4 comprisesmethionine at position +1 to aspartic acid at position +124 (FIG. 2).Alternatively, an extracellular domain of CTLA4 comprises alanine atposition +1 to aspartic acid at position +125 (FIG. 1). Theextracellular domain includes fragments or derivatives of CTLA4 thatbind a B7 molecule (e.g., CD80 and/or CD86).

As used herein a “non-CTLA4 protein sequence” or “non-CTLA4 molecule” isdefined as any molecule that does not bind CD80 and/or CD86 and does notinterfere with the binding of CTLA4 to its target. An example includes,but is not limited to, an immunoglobulin (Ig) constant region or portionthereof. Preferably, the Ig constant region is a human or monkey Igconstant region, e.g., human C(gamma)1, including the hinge, CH2 and CH3regions. The Ig constant region can be mutated to reduce its effectorfunctions (U.S. Pat. Nos. 5,637,481; and 6,090,914).

As used herein, “soluble” refers to any molecule, or fragments andderivatives thereof, not bound or attached to a cell, i.e., circulating.For example, CTLA4, L104EA29YIg, B7 or CD28 can be made soluble byattaching an immunoglobulin (Ig) moiety to the extracellular domain ofCTLA4, B7 or CD28, respectively. Other molecules can be papillomavirusE7 gene product (E7), melanoma-associated antigen p97 (p97) or HIV envprotein (env gp120). Alternatively, a molecule such as CTLA4 can berendered soluble by removing its transmembrane domain. Typically, thesoluble molecules used in the methods of the invention do not include asignal (or leader) sequence.

“CTLA4Ig” is a soluble fusion protein comprising an extracellular domainof CTLA4, or a portion thereof that binds CD80 and/or CD86, joined to anIg tail. A particular embodiment comprises the extracellular domain ofwild type CTLA4 starting at methionine at position +1 and ending ataspartic acid at position +124; or starting at alanine at position −1 toaspartic acid at position +124; a junction amino acid residue glutamineat position +125; and an immunoglobulin portion encompassing glutamicacid at position +126 through lysine at position +357 (FIG. 2). DNAencoding CTLA4Ig was deposited on May 31, 1991 with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209 under the provisions of the Budapest Treaty, and has beenaccorded ATCC accession number ATCC 68629; Linsley, P., et al., 1994Immunity 1:793-80). CTLA4Ig-24, a Chinese Hamster Ovary (CHO) cell lineexpressing CTLA4Ig was deposited on May 31, 1991 with ATCCidentification number CRL-10762). The soluble CTLA4Ig molecules used inthe methods and/or kits of the invention may or may not include a signal(leader) peptide sequence. Typically, in the methods and/or kits of theinvention, the molecules do not include a signal peptide sequence.

As used herein, “soluble CTLA4 molecules” means non-cell-surface-bound(i.e., circulating) CTLA4 molecules (wildtype or mutant) or anyfunctional portion of a CTLA4 molecule that binds B7 including, but notlimited to: CTLA4Ig fusion proteins (e.g., ATCC 68629), wherein theextracellular domain of CTLA4 is fused to an immunoglobulin (Ig) moietyrendering the fusion molecule soluble, or fragments and derivativesthereof; proteins with the extracellular domain of CTLA4 fused or joinedwith a portion of a biologically active or chemically active proteinsuch as the papillomavirus E7 gene product (CTLA4-E7),melanoma-associated antigen p97 (CTLA4-p97) or HIV env protein(CTLA4-env gp120), or fragments and derivatives thereof; hybrid(chimeric) fusion proteins such as CD28/CTLA4Ig, or fragments andderivatives thereof; CTLA4 molecules with the transmembrane domainremoved to render the protein soluble (Oaks, M. K., et al., 2000Cellular Immunology 201:144-153), or fragments and derivatives thereof.“Soluble CTLA4 molecules” also include fragments, portions orderivatives thereof, and soluble CTLA4 mutant molecules having CTLA4binding activity. The soluble CTLA4 molecules used in the methods of theinvention may or may not include a signal (leader) peptide sequence.Typically, in the methods of the invention, the molecules do not includea signal peptide sequence.

As used herein, a “fusion protein” is defined as one or more amino acidsequences joined together using methods well known in the art and asdescribed in U.S. Pat. No. 5,434,131 or 5,637,481. The joined amino acidsequences thereby form one fusion protein.

As used herein a “CTLA4 mutant molecule” is a molecule that can be fulllength CTLA4 or portions thereof (derivatives or fragments) that have amutation or multiple mutations in CTLA4 (preferably in the extracellulardomain of CTLA4) so that it is similar but not identical to the wildtype CTLA4 molecule. CTLA4 mutant molecules bind a B7 molecule (e.g.,either CD80 or CD86, or both). Mutant CTLA4 molecules may include abiologically or chemically active non-CTLA4 molecule therein or attachedthereto. The mutant molecules may be soluble (i.e., circulating) orbound to a surface. CTLA4 mutant molecules can include the entireextracellular domain of CTLA4 or portions thereof, e.g., fragments orderivatives. CTLA4 mutant molecules can be made synthetically orrecombinantly.

As used herein, the term “mutation” is a change in the nucleotide oramino acid sequence of a wild-type polypeptide. The present inventionprovides a mutation or a change in the wild type CTLA4 extracellulardomain. The changes in the wild type CTLA4 sequence include conservativeand non-conservative changes. The change can be an amino acid changewhich includes substitutions, deletions, additions, or truncations. Amutant molecule can have one or more mutations. Mutations in anucleotide sequence may or may not result in a mutation in the aminoacid sequence as is well understood in the art. In that regard, certainnucleotide codons encode the same amino acid. Examples includenucleotide codons CGT, CGG, CGC, and CGA encoding the amino acid,arginine (R); or codons GAT, and GAC encoding the amino acid, asparticacid (D). Thus, a protein can be encoded by one or more nucleic acidmolecules that differ in their specific nucleotide sequence, but stillencode protein molecules having identical sequences. The amino acidcoding sequence is as follows:

One Letter Amino Acid Symbol Symbol Codons Alanine Ala A GCU, GCC, GCA,GCG Cysteine Cys C UGU, UGC Aspartic Acid Asp D GAU, GAC Glutamic AcidGlu E GAA, GAG Phenylalanine Phe F UUU, UUC Glycine Gly G GGU, GGC, GGA,GGG Histidine His H CAU, CAC Isoleucine Ile I AUU, AUC, AUA Lysine Lys KAAA, AAG Leucine Leu L UUA, UUG, CUU, CUC, CUA, CUG Methionine Met M AUGAsparagine Asn N AUU, AAC Proline Pro P CCU, CCC, CCA, CCG Glutamine GlnQ CAA, CAG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Serine Ser S UCU,UCC, UCA, UCG, AGU, AGC Threonine Thr T ACU, ACC, ACA, ACG Valine Val VGUU, GUC, GUA, GUG Tryptophan Trp W UGG Tyrosine Tyr Y UAU, UAC

“L04EA29YIg” is a fusion protein that is a soluble CTLA4 mutant moleculecomprising an extracellular domain of wildtype CTLA4 having amino acidchanges A29Y (a tyrosine amino acid residue substituting for an alanineat position 29) and L104E (a glutamic acid amino acid residuesubstituting for a leucine at position +104), or a portion thereof thatbinds a B7 molecule, joined to an Ig tail (included in FIG. 3; DNAencoding L104EA29YIg was deposited with the American Type CultureCollection on Jun. 20, 2000 and assigned ATCC number PTA-2104). Thesoluble L104EA29YIg molecules used in the methods and/or kits of theinvention may or may not include a signal (leader) peptide sequence.Typically, in the methods and/or kits of the invention, the molecules donot include a signal peptide sequence.

The mutant molecule may have one or more mutations. As used herein, a“non-CTLA4 protein sequence” or “non-CTLA4 molecule” means any proteinmolecule that does not bind B7 and does not interfere with the bindingof CTLA4 to its target. An example includes, but is not limited to, animmunoglobulin (Ig) constant region or portion thereof. Preferably, theIg constant region is a human or monkey Ig constant region, e.g., humanC(gamma)1, including the hinge, CH2 and CH3 regions. The Ig constantregion can be mutated to reduce its effector functions (U.S. Pat. Nos.5,637,481, 5,844,095 and 5,434,131).

As used herein, a “fragment” or “portion” is any part or segment of amolecule e.g. CTLA4 or CD28, preferably the extracellular domain ofCTLA4 or CD28 or a part or segment thereof, that recognizes and bindsits target, e.g., a B7 molecule.

As used herein, “B7” refers to the B7 family of molecules including, butnot limited to, B7-1 (CD80) (Freeman et al, 1989, J Immunol.143:2714-2722, herein incorporated by reference in its entirety), B7-2(CD86) (Freeman et al, 1993, Science 262:909-911 herein incorporated byreference in its entirety; Azuma et al, 1993, Nature 366:76-79 hereinincorporated by reference in its entirety) that may recognize and bindCTLA4 and/or CD28.

As used herein, “CD28” refers to the molecule that recognizes and bindsB7 as described in U.S. Serial No. 5,580,756 and 5,521,288 (hereinincorporated by reference in their entirety).

As used herein, “B7-positive cells” are any cells with one or more typesof B7 molecules expressed on the cell surface.

As used herein, a “derivative” is a molecule that shares sequencesimilarity and activity of its parent molecule. For example, aderivative of CTLA4 includes a soluble CTLA4 molecule having an aminoacid sequence at least 70% similar to the extracellular domain ofwildtype CTLA4, and which recognizes and binds B7 e.g. CTLA4Ig orsoluble CTLA4 mutant molecule L104EA29YIg.

As used herein, to “block” or “inhibit” a receptor, signal or moleculemeans to interfere with the activation of the receptor, signal ormolecule, as detected by an art-recognized test. For example, blockageof a cell-mediated immune response can be detected by determiningreduction of Rheumatic Disease associated symptoms. Blockage orinhibition may be partial or total.

As used herein, “blocking B7 interaction” means to interfere with thebinding of B7 to its ligands, such as CD28 and/or CTLA4, therebyobstructing T-cell and B7-positive cell interactions. Examples of agentsthat block B7 interactions include, but are not limited to, moleculessuch as an antibody (or portion or derivative thereof) that recognizesand binds to the any of CTLA4, CD28 or B7 molecules (e.g. B7-1, B7-2); asoluble form (or portion or derivative thereof) of the molecules such assoluble CTLA4; a peptide fragment or other small molecule designed tointerfere with the cell signal through the CTLA4/CD28/B7-mediatedinteraction. In a preferred embodiment, the blocking agent is a solubleCTLA4 molecule, such as CTLA4Ig (ATCC 68629) or L104EA29YIg (ATCCPTA-2104), a soluble CD28 molecule such as CD28Ig (ATCC 68628), asoluble B7 molecule such as B7Ig (ATCC 68627), an anti-B7 monoclonalantibody (e.g. ATCC HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301,ATCC HB-11341 and monoclonal antibodies as described in by Anderson etal in U.S. Pat. No. 6,113,898 or Yokochi et al., 1982. J. Immun.,128(2):823-827), an anti-CTLA4 monoclonal antibody (e.g. ATCC HB-304,and monoclonal antibodies as described in references 82-83) and/or ananti-CD28 monoclonal antibody (e.g. ATCC HB 11944 and mAb 9.3 asdescribed by Hansen (Hansen et al., 1980. Immunogenetics 10: 247-260) orMartin (Martin et al., 1984. J. Clin. Immun., 4(1):18-22)).

As used herein, “immune system disease” means any disease mediated byT-cell interactions with B7-positive cells including, but not limitedto, autoimmune diseases, graft related disorders and immunoproliferativediseases. Examples of immune system diseases include graft versus hostdisease (GVHD) (e.g., such as may result from bone marrowtransplantation, or in the induction of tolerance), immune disordersassociated with graft transplantation rejection, chronic rejection, andtissue or cell allo- or xenografts, including solid organs, skin,islets, muscles, hepatocytes, neurons. Examples of immunoproliferativediseases include, but are not limited to, psoriasis, T-cell lymphoma,T-cell acute lymphoblastic leukemia, testicular angiocentric T-celllymphoma, benign lymphocytic angiitis, lupus (e.g. lupus erythematosus,lupus nephritis), Hashimoto's thyroiditis, primary myxedema, Graves'disease, pernicious anemia, autoimmune atrophic gastritis, Addison'sdisease, diabetes (e.g. insulin dependent diabetes mellitis, type Idiabetes mellitis, type II diabetes mellitis), good pasture's syndrome,myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia,autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia,idiopathic thrombocytopenia, primary biliary cirrhosis, chronic actionhepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic diseases(e.g. rheumatoid arthritis), polymyositis, scleroderma, and mixedconnective tissue disease.

As used herein, “subject” includes but is not limited to human,non-human primates (e.g., monkey, ape), sheep, rabbit, pig, dog, cat,mouse, or rat.

As used herein, “tissue transplant” is defined as a tissue of all, orpart of, an organ that is transplanted to a recipient subject. Incertain embodiments, the tissue is from one or more solid organs.Examples of tissues or organs include, but are not limited to, skin,heart, lung, pancreas, kidney, liver, bone marrow, pancreatic isletcells, pluripotent stem cells, cell suspensions, and geneticallymodified cells. The tissue can be removed from a donor subject, or canbe grown in vitro. The transplant can be an autograft, isograft,allograft, or xenograft, or a combination thereof.

As used herein, “transplant rejection” is defined as the nearlycomplete, or complete, loss of viable graft tissue from the recipientsubject.

As used herein, “encapsulation” is defined as a process thatimmunoisolates cells and/or cell clusters, which produce and secretetherapeutic substances, e.g. insulin, and to the medical use of theseformulations. The encapsulation process involves the placement of thecells and/or cell clusters within a semipermeable membrane barrier priorto transplantation in order to avoid rejection by the immune system. Themolecular weight cut-off of the encapsulating membrane can be controlledby the encapsulation procedure so as to exclude inward diffusion ofimmunoglobulin and lytic factors of the complement system, but allow thepassage of smaller molecules such as glucose and insulin. Encapsulationpermits the islet cells to respond physiologically to changes in bloodglucose but prevents contact with components of the immune system.Methods of encapsulation of pancreatic islet cells are described in U.S.Pat. No. 6,080,412.

As used herein, “ligand” refers to a molecule that specificallyrecognizes and binds another molecule, for example, a ligand for CTLA4is a CD80 and/or CD86 molecule.

As used herein, “a soluble ligand which recognizes and binds CD80 and/orCD86 antigen” includes ligands such as CTLA4Ig, CD28Ig or other solubleforms of CTLA4 and CD28; recombinant CTLA4 and CD28; mutant CTLA4molecules such as L104EA29YIg; and any antibody molecule, fragmentthereof or recombinant binding protein that recognizes and binds a CD80and/or CD86 antigen. These agents are also considered “immunosuppressiveagents”.

As used herein, “costimulatory pathway” is defined as a biochemicalpathway resulting from interaction of costimulatory signals on T cellsand antigen presenting cells (APCs). Costimulatory signals helpdetermine the magnitude of an immunological response to an antigen. Onecostimulatory signal is provided by the interaction with T cellreceptors CD28 and CTLA4 with CD80 and/or CD86 molecules on APCs.

As used herein, “CD80 and/or CD86” includes B7-1 (also called CD80).B7-2 (also called CD86), B7-3 (also called CD74), and the B7 family,e.g., a combination of B7-1, B7-2, and/or B7-3.

As used herein, “costimulatory blockade” is defined as a protocol ofadministering to a subject, one or more agents that interfere or block acostimulatory pathway, as described above. Examples of agents thatinterfere with the costimulatory blockade include, but are not limitedto, soluble CTLA4, mutant CTLA4, soluble CD28, anti-B7 monoclonalantibodies (mAbs), soluble CD40, and anti-gp39 mAbs. In one embodiment,L104EA29YIg is a preferred agent that interferes with the costimulatoryblockade.

As used herein, “T cell depleted bone marrow” is defined as bone marrowremoved from bone that has been exposed to an anti-T cell protocol. Ananti-T cell protocol is defined as a procedure for removing T cells frombone marrow. Methods of selectively removing T cells are well known inthe art. An example of an anti-T cell protocol is exposing bone marrowto T cell specific antibodies, such as anti-CD3, anti-CD4, anti-CD5,anti-CD8, and anti-CD90 monoclonal antibodies, wherein the antibodiesare cytotoxic to the T cells. Alternatively, the antibodies can becoupled to magnetic particles to permit removal of T cells from bonemarrow using magnetic fields. Another example of an anti-T cell protocolis exposing bone marrow T cells to anti-lymphocyte serum oranti-thymocyte globulin.

As used herein, “tolerizing dose of T cell depleted bone marrow” isdefined as an initial dose of T cell depleted bone marrow that isadministered to a subject for the purpose of inactivating potentialdonor reactive T cells.

As used herein, “engrafting dose of T cell depleted bone marrow” isdefined as a subsequent dose of T cell depleted bone marrow that isadministered to a subject for the purpose of establishing mixedhematopoietic chimerism. The engrafting dose of T cell depleted bonemarrow will accordingly be administered after the tolerizing dose of Tcell depleted bone marrow.

As used herein, “mixed hematopoietic chimerism” is defined as thepresence of donor and recipient blood progenitor and mature cells (e.g.,blood deriving cells) in the absence (or undetectable presence) of animmune response.

As used herein “Donor-recipient pairings” are defined based on moleculartyping using a panel of previously defined major histocompatibiltyalleles (8 class 1 and 12 class II) (Lobashevsky A, et al., TissueAntigens 54:254-263, (1999); Knapp L A, et al., Tissue Antigens50:657-661, (1997); Watkins D. I., Crit. Rev Immunol 15:1-29, (1995)).Pairings maximized disparity at both class I and II loci.

As used herein, “administer” or “administering” to a subject includesbut not limited to intravenous (i.v.) administration, intraperitoneal(i.p.) administration, intramuscular (i.m.) administration, subcutaneousadministration, oral administration, administration by injection, as asuppository, or the implantation of a slow-release device such as aminiosmotic pump, to the subject.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with the reactive agent, retains thereactive agent's biological activity, e.g., binding specificity and isnon-reactive with the subject's immune system. Examples include, but arenot limited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents. Other carriers may alsoinclude sterile solutions, tablets, including coated tablets andcapsules. Typically, such carriers contain excipients, such as starch,milk, sugar, certain types of clay, gelatin, stearic acid or salts,thereof, magnesium or calcium stearate, talc, vegetable fats or oils,gums, glycols, or other known excipients. Such carriers may also includeflavor and color additives or other ingredients. Compositions comprisingsuch carriers are formulated by well-known conventional methods.

As used herein, “immunosuppressive agents” are defined as a compositionhaving one or more types of molecules that prevent the occurrence of animmune response, or weaken a subject's immune system. Preferably, theagents reduce or prevent T cell proliferation. Some agents may inhibit Tcell proliferation by inhibiting interaction of T cells with otherantigen presenting cells (APCs). One example of APCs is B cells.Examples of agents that interfere with T cell interactions with APCs,and thereby inhibit T cell proliferation, include, but are not limitedto, ligands for CD80 and/or CD86 antigens, ligands for CTLA4 antigen,and ligands for CD28 antigen. Examples of ligands for CD80 and/or CD86antigens include, but are not limited to, soluble CTLA4, soluble CTLA4mutant, soluble CD28, or monoclonal antibodies that recognize and bindCD80 and/or CD86 antigens, or fragments thereof. One preferred agent isL104EA29YIg. Ligands for CTLA4 or CD28 antigens include monoclonalantibodies that recognize and bind CTLA4 and/or CD28, or fragmentsthereof. Other ligands for CTLA4 or CD28 include soluble CD80 and/orCD86 molecules, such as CD80 and/or CD86Ig. Persons skilled in the artwill readily understand that other agents or ligands can be used toinhibit the interaction of CD28 with CD80 and/or CD86.

Immunosuppressive agents include, but are not limited to, methotrexate,cyclophosphamide, cyclosporine, cyclosporin A, chloroquine,hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts,D-penicillamine, leflunomide, azathioprine, anakinra, infliximab(REMICADE^(R)), etanercept, TNFα blockers, a biological agent thattargets an inflammatory cytokine, and Non-Steroidal Anti-InflammatoryDrug (NSAIDs). NSAIDs include, but are not limited to acetyl salicylicacid, choline magnesium salicylate, diflunisal, magnesium salicylate,salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen,flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate,naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

Compositions of the Invention

The present invention provides compositions for treating immunediseases, such as diabetes, comprising soluble CTLA4 molecules. Theinvention further provides compositions for inhibiting transplantrejections, e.g. islet cell transplant rejection for treating diabetes.Further, the present invention provides compositions comprising abiological agent that inhibits T-cell function but not T-cell depletionin a human by contacting B7-positive cells in the human with a solubleCTLA4. Examples of soluble CTLA4 include CTLA4Ig and soluble CTLA4mutant molecules such as L104EA29YIg, L104EA29LIg, L104EA29TIg, andL104EA29WIg

CTLA4 molecules, with mutant or wildtype sequences, may be renderedsoluble by deleting the CTLA4 transmembrane segment (Oaks, M. K., etal., 2000 Cellular Immunology 201:144-153).

Alternatively, soluble CTLA4 molecules, with mutant or wildtypesequences, may be fusion proteins, wherein the CTLA4 molecules are fusedto non-CTLA4 moieties such as immunoglobulin (Ig) molecules that renderthe CTLA4 molecules soluble. For example, a CTLA4 fusion protein mayinclude the extracellular domain of CTLA4 fused to an immunoglobulinconstant domain, resulting in the CTLA4Ig molecule (FIG. 2) (Linsley, P.S., et al., 1994 Immunity 1:793-80).

For clinical protocols, it is preferred that the immunoglobulin moietydoes not elicit a detrimental immune response in a subject. Thepreferred moiety is the immunoglobulin constant region, including thehuman or monkey immunoglobulin constant regions. One example of asuitable immunoglobulin region is human Cγ1, including the hinge, CH2and CH3 regions which can mediate effector functions such as binding toFc receptors, mediating complement-dependent cytotoxicity (CDC), ormediate antibody-dependent cell-mediated cytotoxicity (ADCC). Theimmunoglobulin moiety may have one or more mutations therein, (e.g., inthe CH2 domain, to reduce effector functions such as CDC or ADCC) wherethe mutation modulates the binding capability of the immunoglobulin toits ligand, by increasing or decreasing the binding capability of theimmunoglobulin to Fc receptors. For example, mutations in theimmunoglobulin moiety may include changes in any or all its cysteineresidues within the hinge domain, for example, the cysteines atpositions +130, +136, and +139 are substituted with serine (FIG. 24).The immunoglobulin moiety may also include the proline at position +148substituted with a serine, as shown in FIG. 24. Further, the mutationsin the immunoglobulin moiety may include having the leucine at position+144 substituted with phenylalanine, leucine at position +145substituted with glutamic acid, or glycine at position +147 substitutedwith alanine.

Additional non-CTLA4 moieties for use in the soluble CTLA4 molecules orsoluble CTLA4 mutant molecules include, but are not limited to, p97molecule, env gp120 molecule, E7 molecule, and ova molecule (Dash, B. etal. 1994 J. Gen. Virol. 75 (Pt 6):1389-97; Ikeda, T., et al. 1994 Gene138(1-2):193-6; Falk, K., et al. 1993 Cell. Immunol. 150(2):447-52;Fujisaka, K. et al. 1994 Virology 204(2):789-93). Other molecules arealso possible (Gerard, C. et al. 1994 Neuroscience 62(3):721; Byrn, R.et al. 1989 63(10):4370; Smith, D. et al. 1987 Science 238:1704; Lasky,L. 1996 Science 233:209).

The present invention provides soluble CTLA4 molecules including asignal peptide sequence linked to the N-terminal end of theextracellular domain of the CTLA4 portion of the molecule. The signalpeptide can be any sequence that will permit secretion of the mutantmolecule, including the signal peptide from oncostatin M (Malik, et al.,1989 Molec. Cell. Biol. 9: 2847-2853), or CD5 (Jones, N. H. et al., 1986Nature 323:346-349), or the signal peptide from any extracellularprotein. The soluble CTLA4 molecule of the invention can include theoncostatin M signal peptide linked at the N-terminal end of theextracellular domain of CTLA4, and the human immunoglobulin molecule(e.g., hinge, CH2 and CH3) linked to the C-terminal end of theextracellular domain (wildtype or mutated) of CTLA4. This moleculeincludes the oncostatin M signal peptide encompassing an amino acidsequence having methionine at position −26 through alanine at position−1, the CTLA4 portion encompassing an amino acid sequence havingmethionine at position +1 through aspaitic acid at position +124, ajunction amino acid residue glutamine at position +125, and theimmunoglobulin portion encompassing an amino acid sequence havingglutamic acid at position +126 through lysine at position +357.

In one embodiment, the soluble CTLA4 mutant molecules of the invention,comprising the mutated CTLA4 sequences described infra, are fusionmolecules comprising human IgC(gamma)1 (i.e. IgCγ1) moieties fused tothe mutated CTLA4 fragments. The soluble CTLA4 mutant molecules cancomprise one or more mutations (e.g., amino acid substitutions,deletions, or insertions) in the extracellular domain of CTLA4.

For example, the soluble CTLA4 mutant molecules can include a mutationor mutations within or in close proximity to the region encompassed byserine at position +25 through arginine at position +33 (e.g., S25-R33,using standard single-letter amino acid symbols). The mutant CTLA4molecules can include an amino acid substitution at any one or more ofthe following positions: S25, P26, G27, K28, A29, T30, E31, or R33.

In another embodiment, the soluble CTLA4 mutant molecules can include amutation or mutations within or in close proximity to the regionencompassed by glutamic acid at position +95 to glycine at position +107(e.g., E95-G107) The mutant CTLA4 molecules can include an amino acidsubstitution at any one or more of the following positions: K93, L96,M97, Y98, P99, P100, P101, Y102, Y103, L104, G105, 1106, and G107.

Additionally, the invention provides soluble CTLA4 mutant moleculeshaving a mutation or mutations within or in close proximity to theregion encompassed by asparagine +108 to isoleucine at position +115(e.g., N108-1115). The mutant CTLA4 molecule can include an amino acidsubstitution at any one or more of the following positions: L104, G105,1106, G107, Q111, Y113, or 1115.

In one embodiment, the soluble CTLA4 mutant molecules comprise IgCγ1fused to a CTLA4 fragment comprising a single-site mutation in theextracellular domain. The extracellular domain of CTLA4 comprisesmethionine at position +1 through aspartic acid at position +124 (e.g.,FIG. 1). The extracellular portion of the CTLA4 can comprise alanine atposition −1 through aspartic acid at position +124 (e.g., FIG. 1).Examples of single-site mutations include the following wherein theleucine at position +104 is changed to any other amino acid:

Single-site mutant: Codon change: L104EIg Glutamic acid GAG L104SIgSerine AGT L104TIg Threonine ACG L104AIg Alanine GCG L104WIg TryptophanTGG L104QIg Glutamine CAG L104KIg Lysine AAG L104RIg Arginine CGGL104GIg Glycine GGG

Further, the invention provides mutant molecules having theextracellular domain of CTLA4 with two mutations, fused to an Ig Cγ1moiety. Examples include the following wherein the leucine at position+104 is changed to another amino acid (e.g. glutamic acid) and theglycine at position +105, the serine at position +25, the threonine atposition +30 or the alanine at position +29 is changed to any otheramino acid:

Double-site mutants: Codon change: L104EG105FIg Phenylalanine TTCL104EG105WIg Tryptophan TGG L104EG105LIg Leucine CTT L104ES25RIgArginine CGG L104ET30GIg Glycine GGG L104ET30NIg Asparagine AATL104EA29YIg Tyrosine TAT L104EA29LIg Leucine TTG L104EA29TIg ThreonineACT L104EA29WIg Tryptophan TGG

Further still, the invention provides mutant molecules having theextracellular domain of CTLA4 comprising three mutations, fused to an IgCγ1 moiety. Examples include the following wherein the leucine atposition +104 is changed to another amino acid (e.g. glutamic acid), thealanine at position +29 is changed to another amino acid (e.g. tyrosine)and the serine at position +25 is changed to another amino acid:

Triple-site Mutants: Codon changes: L104EA29YS25KIg Lysine AAAL104EA29YS25KIg Lysine AAG L104EA29YS25NIg Asparagine AACL104EA29YS25RIg Arginine CGG

Soluble CTLA4 mutant molecules may have a junction amino acid residuewhich is located between the CTLA4 portion and the Ig portion of themolecule. The junction amino acid can be any amino acid, includingglutamine. The junction amino acid can be introduced by molecular orchemical synthesis methods known in the art.

The invention provides soluble CTLA4 mutant molecules comprising asingle-site mutation in the extracellular domain of CTLA4 such asL104EIg (as included in FIG. 19) or L104SIg, wherein L104EIg and L104SIgare mutated in their CTLA4 sequences so that leucine at position +104 issubstituted with glutamic acid or serine, respectively. The single-sitemutant molecules further include CTLA4 portions encompassing methionineat position +1 through aspartic acid at position +124, a junction aminoacid residue glutamine at position +125, and an immunoglobulin portionencompassing glutamic acid at position +126 through lysine at position+357. The immunoglobulin portion of the mutant molecule may also bemutated so that the cysteines at positions+130, +136, and +139 aresubstituted with serine, and the proline at position +148 is substitutedwith serine. Alternatively, the single-site soluble CTLA4 mutantmolecule may have a CTLA4 portion encompassing alanine at position −1through aspartic acid at position +124.

The invention provides soluble CTLA4 mutant molecules comprising adouble-site mutation in the extracellular domain of CTLA4, such asL104EA29YIg, L104EA29LIg, L104EA29TIg or L104EA29WIg, wherein leucine atposition +104 is substituted with a glutamic acid and alanine atposition +29 is changed to tyrosine, leucine, threonine and tryptophan,respectively. The sequences for L104EA29YIg, L104EA29LIg, L104EA29TIgand L104EA29WIg, starting at methionine at position +1 and ending withlysine at position +357, plus a signal (leader) peptide sequence areincluded in the sequences as shown in FIGS. 3 and 20-22 respectively.The double-site mutant molecules further comprise CTLA4 portionsencompassing methionine at position +1 through aspartic acid at position+124, a junction amino acid residue glutamine at position +125, and animmunoglobulin portion encompassing glutamic acid at position +126through lysine at position +357. The immunoglobulin portion of themutant molecule may also be mutated, so that the cysteines atpositions+130, +136, and +139 are substituted with serine, and theproline at position +148 is substituted with serine. Alternatively,these mutant molecules can have a CTLA4 portion encompassing alanine atposition −1 through aspartic acid at position +124.

The invention provides soluble CTLA4 mutant molecules comprising adouble-site mutation in the extracellular domain of CTLA4, such asL104EG105FIg, L104EG105WIg and L104EG105LIg, wherein leucine at position+104 is substituted with a glutamic acid and glycine at position +105 issubstituted with phenylalanine, tryptophan and leucine, respectively.The double-site mutant molecules further comprise CTLA4 portionsencompassing methionine at position +1 through aspartic acid at position+124, a junction amino acid residue glutamine at position +125, and animmunoglobulin portion encompassing glutamic acid at position +126through lysine at position +357. The immunoglobulin portion of the mayalso be mutated, so that the cysteines at positions +130, +136, and +139are substituted with serine, and the proline at position +148 issubstituted with serine. Alternatively, these mutant molecules can havea CTLA4 portion encompassing alanine at position −1 through asparticacid at position +124.

The invention provides L104ES25RIg which is a double-site mutantmolecule including a CTLA4 portion encompassing methionine at position+1 through aspartic acid at position +124, a junction amino acid residueglutamine at position +125, and the immunoglobulin portion encompassingglutamic acid at position +126 through lysine at position +357. Theportion having the extracellular domain of CTLA4 is mutated so thatserine at position +25 is substituted with arginine, and leucine atposition +104 is substituted with glutamic acid. Alternatively,L104ES25RIg can have a CTLA4 portion encompassing alanine at position −1through aspartic acid at position +124.

The invention provides soluble CTLA4 mutant molecules comprising adouble-site mutation in the extracellular domain of CTLA4, such asL104ET30GIg and L104ET30NIg, wherein leucine at position +104 issubstituted with a glutamic acid and threonine at position +30 issubstituted with glycine and asparagine, respectively. The double-sitemutant molecules further comprise CTLA4 portions encompassing methionineat position +1 through aspartic acid at position +124, a junction aminoacid residue glutamine at position +125, and an immunoglobulin portionencompassing glutamic acid at position +126 through lysine at position+357. The immunoglobulin portion of the mutant molecule may also bemutated, so that the cysteines at positions +130, +136, and +139 aresubstituted with serine, and the proline at position +148 is substitutedwith serine. Alternatively, these mutant molecules can have a CTLA4portion encompassing alanine at position −1 through aspartic acid atposition +124.

The invention provides soluble CTLA4 mutant molecules comprising atriple-site mutation in the extracellular domain of CTLA4, such asL104EA29YS25KIg, L104EA29YS25NIg, L104EA29YS25RIg, wherein leucine atposition +104 is substituted with a glutamic acid, alanine at position+29 is changed to tyrosine and serine at position +25 is changed tolysine, asparagine and arginine, respectively. The triple-site mutantmolecules further comprise CTLA4 portions encompassing methionine atposition +1 through aspartic acid at position +124, a junction aminoacid residue glutamine at position +125, and an immunoglobulin portionencompassing glutamic acid at position +126 through lysine at position+357. The immunoglobulin portion of the mutant molecule may also bemutated, so that the cysteines at positions+130, +136, and +139 aresubstituted with serine, and the proline at position +148 is substitutedwith serine. Alternatively, these mutant molecules can have a CTLA4portion encompassing alanine at position −1 through aspartic acid atposition +124.

Additional embodiments of soluble CTLA4 mutant molecules includechimeric CTLA4/CD28 homologue mutant molecules that bind a B7 (Peach, R.J., et al., 1994 J Exp Med 180:2049-2058). Examples of these chimericCTLA4/CD28 mutant molecules include HS1, HS2, HS3, HS4, HS5, HS6, HS4A,HS4B, HS7, HS8, HS9, HS10, HS11, HS12, HS13 and HS14 (U.S. Pat. No.5,773,253)

Preferred embodiments of the invention are soluble CTLA4 molecules suchas CTLA4Ig (as shown in FIG. 2, starting at methionine at position +1and ending at lysine at position +357) and soluble CTLA4 mutantL104EA29YIg (as shown in FIG. 3, starting at methionine at position +1and ending at lysine at position +357).

The invention further provides nucleic acid molecules comprisingnucleotide sequences encoding the amino acid sequences corresponding tothe soluble CTLA4 molecules of the invention. In one embodiment, thenucleic acid molecule is a DNA (e.g., cDNA) or a hybrid thereof. DNAencoding CTLA4Ig (FIG. 2) was deposited on May 31, 1991 with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209 and has been accorded ATCC accession numberATCC 68629. DNA encoding L104EA29YIg (sequence included in FIG. 3) wasdeposited on Jun. 19, 2000 with ATCC and has been accorded ATCCaccession number PTA-2104. Alternatively, the nucleic acid molecules areRNA or a hybrid thereof.

CTLA4 Hybrids

The present invention provides soluble CTLA4 mutant molecules comprisingat least the extracellular domain of CTLA4 or portions thereof that bindCD80 and/or CD86. The extracellular portion of CTLA4 comprisesmethionine at position +1 through aspartic acid at position +124 (e.g.,FIG. 1). The extracellular portion of the CTLA4 can comprise alanine atposition −1 through aspartic acid at position +124 (e.g., FIG. 1). Theextracellular portion of the CTLA4 can comprise glutamic acid atposition +95 through cysteine at position +120. The extracellularportion of the CTLA4 can comprise methionine at position +1 throughcysteine at position +21 and glutamic acid at position +95 throughaspartic acid at position +122. The extracellular portion of the CTLA4can comprise methionine at position +1 through tyrosine at position +23and valine at position +32 through aspartic acid at position +122. Theextracellular portion of the CTLA4 can comprise alanine at position +24through glutamic acid at position +31 and glutamic acid at position +95through aspartic acid at position +122. The extracellular portion of theCTLA4 can comprise alanine at position +24 through glutamic acid atposition +31 and glutamic acid at position +95 through isoleucine atposition +112. The extracellular portion of the CTLA4 can comprisealanine at position +24 through glutamic acid at position +31 andtyrosine at position +113 through aspartic acid at position +122. Theextracellular portion of the CTLA4 can comprise alanine at position +50through glutamic acid at position +57 and glutamic acid at position +95through aspartic acid at position +122. The extracellular portion of theCTLA4 can comprise alanine at position +24 through glutamic acid atposition +31; alanine at position +50 through glutamic acid at position+57; and glutamic acid at position +95 through aspartic acid at position+122. The extracellular portion of the CTLA4 can comprise alanine atposition +50 through glutamic acid at position +57 and glutamic acid atposition +95 through isoleucine at position +112. The extracellularportion of the CTLA4 can comprise alanine at position +24 throughglutamic acid at position +31; alanine at position +50 through glutamicacid at position +57; and glutamic acid at position +95 through asparticacid at position +122. The extracellular portion of CTLA4 can comprisealanine at position +24 through valine at position +94. Theextracellular portion of CTLA4 can comprise alanine at position −1through cysteine at position. +21. The extracellular portion of CTLA4can comprise methionine at position +1 through cysteine at position +21.The extracellular portion of CTLA4 can comprise glutamic acid atposition +95 through aspartic acid at position +122. The extracellularportion of CTLA4 can comprise alanine at position −1 through valine atposition +94. The extracellular portion of CTLA4 can comprise methionineat position +1 through valine at position +94. The extracellular portionof CTLA4 can comprise alanine at position +24 through glutamic acid atposition +31. The extracellular portion of CTLA4 can comprise alanine atposition −1 through tyrosine at position +23. The extracellular portionof CTLA4 can comprise methionine at position +1 through tyrosine atposition +23. The extracellular portion of CTLA4 can comprise valine atposition +32 through aspartic acid at position +122. The extracellularportion of CTLA4 can comprise tyrosine at position +113 through asparticacid at position +122. The extracellular portion of CTLA4 can compriseglutamic acid at position +95 through isoleucine at position +112. Theextracellular portion of CTLA4 can comprise alanine at position +50through glutamic acid at position +57.

Methods for Producing the Molecules of the Invention

Expression of CTLA4 mutant molecules can be in prokaryotic cells.Prokaryotes most frequently are represented by various strains ofbacteria. The bacteria may be a gram positive or a gram negative. Othermicrobial strains may also be used.

Nucleotide sequences encoding CTLA4 mutant molecules can be insertedinto a vector designed for expressing foreign sequences in prokaryoticcells such as E. coli. These vectors can include commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences, include such commonly usedpromoters as the beta-lactamase (penicillinase) and lactose (lac)promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan(trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res.8:4057) and the lambda derived P_(L) promoter and N-gene ribosomebinding site (Shimatake, et al., (1981) Nature 292:128).

Such expression vectors will also include origins of replication andselectable markers, such as a beta-lactamase or neomycinphosphotransferase gene conferring resistance to antibiotics, so thatthe vectors can replicate in bacteria and cells carrying the plasmidscan be selected for when grown in the presence of antibiotics, such asampicillin or kanamycin.

The expression plasmid can be introduced into prokaryotic cells via avariety of standard methods, including but not limited to CaCl₂-shock(Cohen, (1972) Proc. Natl. Acad. Sci. USA 69:2110, and Sambrook et al.(eds.), “Molecular Cloning: A Laboratory Manual”, 2nd Edition, ColdSpring Harbor Press, (1989)) and electroporation.

In accordance with the practice of the invention, eukaryotic cells arealso suitable host cells. Examples of eukaryotic cells include anyanimal cell, whether primary or immortalized, yeast (e.g., Saccharomycescerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), and plantcells. Myeloma, COS and CHO cells are examples of animal cells that maybe used as hosts. Particular CHO cells include, but are not limited to,DG44 (Chasin, et al., 1986 Som. Cell. Molec. Genet. 12:555-556; Kolkekar1997 Biochemistry 36:10901-10909), CHO-K1 (ATCC No. CCL-61), CHO-K1Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR,Salisbury, Wiltshire, LK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B(GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR,Salisbury, Wiltshire, UK), and RR-CHOK1 designated ECACC 92052129 (CAMR,Salisbury, Wiltshire, UK). Exemplary plant cells include tobacco (wholeplants, cell culture, or callus), corn, soybean, and rice cells. Corn,soybean, and rice seeds are also acceptable.

Nucleotide sequences encoding the CTLA4 mutant molecules can also beinserted into a vector designed for expressing foreign sequences in aeukaryotic host. The regulatory elements of the vector can varyaccording to the particular eukaryotic host. The nucleic acid moleculethat encodes L104EA29YIg is contained in pD16 L104EA29YIg and wasdeposited on Jun. 19, 2000 with the American Type Culture Collection(ATCC), 10801 University Blvd., Manasas, Va. 20110-2209 (ATCC No.PTA-2104). The pD16 L104EA29YIg vector is a derivative of the pcDNA3vector (INVITROGEN).

Commonly used eukaryotic control sequences for use in expression vectorsinclude promoters and control sequences compatible with mammalian cellssuch as, for example, CMV promoter (CDM8 vector) and avian sarcoma virus(ASV) (πLN vector). Other commonly used promoters include the early andlate promoters from Simian Virus 40 (SV40) (Fiers, et al., (1973) Nature273:113), or other viral promoters such as those derived from polyoma,Adenovirus 2, and bovine papilloma virus. An inducible promoter, such ashMTII (Karin, et al., (1982) Nature 299:797-802) may also be used.

Vectors for expressing CTLA4 mutant molecules in eukaryotes may alsocarry sequences called enhancer regions. These are important inoptimizing gene expression and are found either upstream or downstreamof the promoter region.

Examples of expression vectors for eukaryotic host cells include, butare not limited to, vectors for mammalian host cells (e.g., BPV-1, pHyg,pRSV, pSV2, pTK2 (Maniatis); pIRES (Clontech); pRc/CMV2, pRc/RSV, pSFV1(Life Technologies); pVPakc Vectors, pCMV vectors, pSG5 vectors(Stratagene)), retroviral vectors (e.g., pFB vectors (Stratagene)),pcDNA-3 (Lnvitrogen) or modified forms thereof, adenoviral vectors;Adeno-associated virus vectors, baculovirus vectors, yeast vectors(e.g., pESC vectors (Stratagene)).

Nucleotide sequences encoding CTLA4 mutant molecules can integrate intothe genome of the eukaryotic host cell and replicate as the host genomereplicates. Alternatively, the vector carrying CTLA4 mutant moleculescan contain origins of replication allowing for extrachromosomalreplication.

For expressing the nucleotide sequences in Saccharomyces cerevisiae, theorigin of replication from the endogenous yeast plasmid, the 2μ circlecan be used. (Broach, (1983) Meth. Enz. 101:307). Alternatively,sequences from the yeast genome capable of promoting autonomousreplication can be used (see, for example, Stinchcomb et al., (1979)Nature 282:39); Tschemper et al., (1980) Gene 10:157; and Clarke et al.,(1983) Meth. Enz. 101:300).

Transcriptional control sequences for yeast vectors include promotersfor the synthesis of glycolytic enzymes (Hess et al., (1968) J. Adv.Enzyme Reg. 7:149; Holland et al., (1978) Biochemistry 17:4900).Additional promoters known in the art include the CMV promoter providedin the CDM8 vector (Toyama and Okayama, (1990) FEBS 268:217-221); thepromoter for 3-phosphoglycerate kinase (Hitzeman et al., (1980) J. Biol.Chem. 255:2073), and those for other glycolytic enzymes.

Other promoters are inducible because they can be regulated byenvironmental stimuli or the growth medium of the cells. These induciblepromoters include those from the genes for heat shock proteins, alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, enzymes associatedwith nitrogen catabolism, and enzymes responsible for maltose andgalactose utilization.

Regulatory sequences may also be placed at the 3′ end of the codingsequences. These sequences may act to stabilize messenger RNA. Suchterminators are found in the 3′ untranslated region following the codingsequences in several yeast-derived and mammalian genes.

Exemplary vectors for plants and plant cells include, but are notlimited to, Agrobacterium T_(i) plasmids, cauliflower mosaic virus(CaMV), and tomato golden mosaic virus (TGMV).

General aspects of mammalian cell host system transformations have beendescribed by Axel (U.S. Pat. No. 4,399,216 issued Aug. 16, 1983).Mammalian cells can be transformed by methods including but not limitedto, transfection in the presence of calcium phosphate, microinjection,electroporation, or via transduction with viral vectors. Methods forintroducing foreign DNA sequences into plant and yeast genomes include(1) mechanical methods, such as microinjection of DNA into single cellsor protoplasts, vortexing cells with glass beads in the presence of DNA,or shooting DNA-coated tungsten or gold spheres into cells orprotoplasts; (2) introducing DNA by making cell membranes permeable tomacromolecules through polyethylene glycol treatment or subjection tohigh voltage electrical pulses (electroporation); or (3) the use ofliposomes (containing cDNA) which fuse to cell membranes.

Expression of CTLA4 mutant molecules can be detected by methods known inthe art. For example, the mutant molecules can be detected by Coomassiestaining SDS-PAGE gels and immunoblotting using antibodies that bindCTLA4. Protein recovery can be performed using standard proteinpurification means, e.g., affinity chromatography or ion-exchangechromatography, to yield substantially pure product (R. Scopes in:“Protein Purification, Principles and Practice”, Third Edition,Springer-Verlag (1994)).

The invention further provides soluble CTLA4 mutant protein moleculesproduced by the methods herein.

CTLA4IG Codon-Based Mutagenesis

In one embodiment, site-directed mutagenesis and a novel screeningprocedure were used to identify several mutations in the extracellulardomain of CTLA4 that improve binding avidity for CD86. In thisembodiment, mutations were carried out in residues in the regions of theextracellular domain of CTLA4 from serine 25 to arginine 33, the C′strand (alanine 49 and threonine 51), the F strand (lysine 93, glutamicacid 95 and leucine 96), and in the region from methionine 97 throughtyrosine 102, tyrosine 103 through glycine 107 and in the G strand atpositions glutamine 111, tyrosine 113 and isoleucine 115. These siteswere chosen based on studies of chimeric CD28/CTLA4 fusion proteins(Peach et al., J. Exp. Med. 1994, 180:2049-2058), and on a modelpredicting which amino acid residue side chains would be solventexposed, and a lack of amino acid residue identity or homology atcertain positions between CD28 and CTLA4. Also, any residue which isspatially in close proximity (5 to 20 Angstrom Units) to the identifiedresidues is considered part of the present invention.

To synthesize and screen soluble CTLA4 mutant molecules with alteredaffinities for CD80 and/or CD86, a two-step strategy was adopted. Theexperiments entailed first generating a library of mutations at aspecific codon of an extracellular portion of CTLA4 and then screeningthese by BIAcore analysis to identify mutants with altered reactivity toCD80 or CD86. The Biacore assay system (Pharmacia, Piscataway, N.J.)uses a surface plasmon resonance detector system that essentiallyinvolves covalent binding of either CD80Ig or CD86Ig to a dextran-coatedsensor chip which is located in a detector. The test molecule can thenbe injected into the chamber containing the sensor chip and the amountof complementary protein that binds can be assessed based on the changein molecular mass which is physically associated with the dextran-coatedside of the sensor chip; the change in molecular mass can be measured bythe detector system.

Pharmaceutical Compositions of the Invention

The invention includes pharmaceutical compositions for use in thetreatment of immune system diseases comprising pharmaceuticallyeffective amounts of soluble CTLA4 mutant molecules. In certainembodiments, the immune system diseases are mediated by CD28- and/orCTLA4-positive cell interactions with CD80 and/or CD86 positive cells.The soluble CTLA4 molecules are preferably soluble CTLA4 molecules withwildtype sequence and/or soluble CTLA4 molecules having one or moremutations in the extracellular domain of CTLA4. The pharmaceuticalcomposition can include soluble CTLA4 or CTLA4 mutant protein moleculesand/or nucleic acid molecules, and/or vectors encoding the molecules. Ina preferred embodiment, the soluble CTLA4 mutant molecule has the aminoacid sequence of the extracellular domain of CTLA4 as shown in eitherFIG. 3 (L104EA29Y). Even more preferably, the soluble CTLA4 mutantmolecule is L104EA29YIg as disclosed herein shown in FIG. 3. Thecompositions may additionally include other therapeutic agents,including, but not limited to, immunosuppressive agents, NSAIDs,corticosteroids, glucococoticoids, drugs, toxins, enzymes, antibodies,or conjugates.

An embodiment of the pharmaceutical composition comprises an effectiveamount of a soluble CTLA4 molecule alone or in combination with aneffective amount of at least one other therapeutic agent, including animmunosuppressive agent, or NSAID.

Effective amounts of soluble CTLA4 in the pharmaceutical composition canrange about 0.1 to 100 mg/kg weight of the subject. In anotherembodiment, the effective amount can be an amount about 0.5 to 5 mg/kgweight of a subject, about 5 to 10 mg/kg weight of a subject, about 10to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of asubject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kgweight of a subject, about 30 to 35 mg/kg weight of a subject, about 35to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject,about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight ofa subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject,about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight ofa subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, orabout 95 to 100 mg/kg weight of a subject. In an embodiment, theeffective amount is 2 mg/kg weight of a subject. In another embodiment,the effective amount is 10 mg/kg weight of a subject. In an embodiment,the effective amount of a soluble CTLA4 molecule is 2 mg/kg weight of asubject. In an embodiment, the effective amount of a soluble CTLA4molecule is 10 mg/kg weight of a subject.

The amount of an immunosuppressive agent administered to a subjectvaries depending on several factors including the efficacy of the drugon a specific subject and the toxicity (i.e. the tolerability) of a drugto a specific subject.

Methotrexate is commonly administered in an amount about 0.1 to 40 mgper week with a common dosage ranging about 5 to 30 mg per week.Methotrexate may be administered to a subject in various increments:about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week,about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week,about 30 to 35 mg/week, or about 35 to 40 mg/week. In one embodiment, aneffective amount of an immunosuppressive agent, including methotrexate,is an amount about 10 to 30 mg/week.

Effective amounts of methotrexate range about 0.1 to 40 mg/week. In oneembodiment, the effective amount is ranges about 0.1 to 5 mg/week, about5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, orabout 35 to 40 mg/week. In one embodiment, methotrexate is administeredin an amount ranging about 10 to 30 mg/week.

Cyclophosphamide, an alkylating agent, may be administered in dosagesranging about 1 to 10 mg/kg body weight per day.

Cyclosporine (e.g. NEORAL^(R)) also known as Cyclosporin A, is commonlyadministered in dosages ranging from about 1 to 10 mg/kg body weight perday. Dosages ranging about 2.5 to 4 mg per body weight per day arecommonly used.

Chloroquine or hydroxychloroquine (e.g. PLAQUENIL^(R)), is commonlyadministered in dosages ranging about 100 to 1000 mg daily. Preferreddosages range about 200-600 mg administered daily.

Sulfasalazine (e.g., AZULFIDINE EN-tabs^(R)) is commonly administered inamounts ranging about 50 to 5000 mg per day, with a common dosage ofabout 2000 to 3000 mg per day for adults. Dosages for children arecommonly about 5 to 100 mg/kg of body weight, up to 2 grams per day.

Gold salts are formulated for two types of administration: injection ororal. Injectable gold salts are commonly prescribed in dosages about 5to 100 mg doses every two to four weeks. Orally administered gold saltsare commonly prescribed in doses ranging about 1 to 10 mg per day.

D-penicillamine or penicillamine (CUPRIMINE^(R)) is commonlyadministered in dosages about 50 to 2000 mg per day, with preferreddosages about 125 mg per day up to 1500 mg per day.

Azathioprine is commonly administered in dosages of about 10 to 250 mgper day. Preferred dosages range about 25 to 200 mg per day.

Anakinra (e.g. KINERET^(R)) is an interleukin-1 receptor antagonist. Acommon dosage range for anakinra is about 10 to 250 mg per day, with arecommended dosage of about 100 mg per day.

Infliximab (REMICADE^(R)) is a chimeric monoclonal antibody that bindsto tumor necrosis factor alpha (TNFα). Infliximab is commonlyadministered in dosages about 1 to 20 mg/kg body weight every four toeight weeks. Dosages of about 3 to 10 mg/kg body weight may beadministered every four to eight weeks depending on the subject.

Etanercept (e.g. ENBREL^(R)) is a dimeric fusion protein that binds thetumor necrosis factor (TNF) and blocks its interactions with TNFreceptors. Commonly administered dosages of etanercept are about 10 to100 mg per week for adults with a preferred dosage of about 50 mg perweek. Dosages for juvenile subjects range about 0.1 to 50 mg/kg bodyweight per week with a maximum of about 50 mg per week.

Leflunomide (ARAVA^(R)) is commonly administered at dosages about 1 and100 mg per day. A common daily dosage is about 10 to 20 mg per day.

The pharmaceutical compositions also preferably include suitablecarriers and adjuvants which include any material which when combinedwith the molecule of the invention (e.g., a soluble CTLA4 mutantmolecule, e.g., L104EA29YIg) retains the molecule's activity and isnon-reactive with the subject's immune system. Examples of suitablecarriers and adjuvants include, but are not limited to, human serumalbumin; ion exchangers; alumina; lecithin; buffer substances, such asphosphates; glycine; sorbic acid; potassium sorbate; and salts orelectrolytes, such as protamine sulfate. Other examples include any ofthe standard pharmaceutical carriers such as a phosphate buffered salinesolution; water; emulsions, such as oil/water emulsion; and varioustypes of wetting agents. Other carriers may also include sterilesolutions; tablets, including coated tablets and capsules. Typicallysuch carriers contain excipients such as starch, milk, sugar, certaintypes of clay, gelatin, stearic acid or salts thereof, magnesium orcalcium stearate, talc, vegetable fats or oils, gums, glycols, or otherknown excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well known conventional methods. Such compositions mayalso be formulated within various lipid compositions, such as, forexample, liposomes as well as in various polymeric compositions, such aspolymer microspheres.

The pharmaceutical compositions of the invention can be administeredusing conventional modes of administration including, but not limitedto, intravenous (i.v.) administration, intraperitoneal (i.p.)administration, intramuscular (i.m.) administration, subcutaneousadministration, oral administration, administration as a suppository, oras a topical contact, or the implantation of a slow-release device suchas a miniosmotic pump, to the subject.

The pharmaceutical compositions of the invention may be in a variety ofdosage forms, which include, but are not limited to, liquid solutions orsuspensions, tablets, pills, powders, suppositories, polymericmicrocapsules or microvesicles, liposomes, and injectable or infusiblesolutions. The preferred form depends upon the mode of administrationand the therapeutic application.

The most effective mode of administration and dosage regimen for thecompositions of this invention depends upon the severity and course ofthe disease, the patient's health and response to treatment and thejudgment of the treating physician. Accordingly, the dosages of thecompositions should be titrated to the individual patient.

The soluble CTLA4 mutant molecules may be administered to a subject inan amount and for a time (e.g. length of time and/or multiple times)sufficient to block endogenous B7 (e.g., CD80 and/or CD86) moleculesfrom binding their respective ligands, in the subject. Blockage ofendogenous B7/ligand binding thereby inhibits interactions betweenB7-positive cells (e.g., CD80- and/or CD86-positive cells) with CD28-and/or CTLA4-positive cells. Dosage of a therapeutic agent is dependentupon many factors including, but not limited to, the type of tissueaffected, the type of autoimmune disease being treated, the severity ofthe disease, a subject's health, and a subject's response to thetreatment with the agents. Accordingly, dosages of the agents can varydepending on the subject and the mode of administration. The solubleCTLA4 mutant molecules may be administered in an amount between 0.1 to20.0 mg/kg weight of the patient/day, preferably between 0.5 to 10.0mg/kg/day. Administration of the pharmaceutical compositions of theinvention can be performed over various times. In one embodiment, thepharmaceutical composition of the invention can be administered for oneor more hours. In addition, the administration can be repeated dependingon the severity of the disease as well as other factors as understood inthe art.

Methods of the Invention

The present invention provides methods for treating immune systemdiseases and auto-immune diseases in a subject comprising administeringto the subject an effective amount of a soluble CTLA4 or a CTLA4 mutantmolecule which binds CD80 and/or CD86 molecules on CD80 and/orCD86-positive cells so as to inhibit binding of CD80 and/or CD86 toCTLA4 and/or CD28. The methods comprise administering a therapeuticcomposition, comprising soluble CTLA4 or CTLA4 mutant molecules of theinvention, to a subject in an amount effective to relieve at least oneof the symptoms associated with immune system diseases. Additionally,the invention may provide long-term therapy for immune system diseasesby blocking the T-cell/B7-positive cell interactions, thereby blockingT-cell activation/stimulation by co-stimulatory signals such as B7binding to CD28, leading to induction of T-cell anergy or tolerance.

The soluble CTLA4 or CTLA4 mutant molecules of the invention exhibitinhibitory properties in vivo. Under conditions where T-cell/B7-positivecell interactions, for example T cell/B cell interactions, are occurringas a result of contact between T cells and B7-positive cells, binding ofintroduced CTLA4 molecules to react to B7-positive cells, for example Bcells, may interfere, i.e., inhibit, the T cell/B7-positive cellinteractions resulting in regulation of immune responses. Inhibition ofT cell responses by administering a soluble CTLA4 molecule may also beuseful for treating autoimmune disorders. Many autoimmune disordersresult from inappropriate activation of T cells that are reactiveagainst autoantigens, and which promote the production of cytokines andautoantibodies that are involved in the pathology of the disease.Administration of L104EA2-9YIg molecule in a subject suffering from orsusceptible to an autoimmune disorder may prevent the activation ofautoreactive T cells and may reduce or eliminate disease symptoms. Thismethod may also comprise administering to the subject L104EA29YIgmolecule of the invention, alone or together, with additional ligands,such as those reactive with IL-2, IL-4, or γ-interferon.

The invention provides methods for regulating immune responses. Immuneresponses may be down-regulated (reduced) by the soluble CTLA4 or CTLA4mutant molecules of the invention may be by way of inhibiting orblocking an immune response already in progress or may involvepreventing the induction of an immune response. The soluble CTLA4 orCTLA4 mutant molecules of the invention may inhibit the functions ofactivated T cells, such as T lymphocyte proliferation and cytokinesecretion, by suppressing T cell responses or by inducing specifictolerance in T cells, or both. Further, the soluble CTLA4 or CTLA4mutant molecules of this invention, interfering with the CTLA4/CD28/B7pathway may inhibit T-cell proliferation and/or cytokine secretion, andthus result in reduced tissue destruction and induction of T-cellunresponsiveness or anergy.

The invention further provides methods for inhibiting rejection of organor tissue transplants in subjects comprising administering an effectiveamount of at least one soluble CTLA4 or CTLA4 molecule, e.g.,L104EA29YIg, to the subject before, during and/or after transplantation.In another embodiment, the method of the invention include administeringto a subject at least one soluble CTLA4 or a CTLA4 mutant molecule incombination with at least one other therapeutic agent, including, butnot limited to a drug, a toxin, an enzyme, an antibody, or a conjugate.

The organ or tissue transplant can be from any type of organ or tissueamenable to transplantation. In one embodiment, the transplanted tissuecan be a pancreatic tissue. In a preferred embodiment, the transplanttissue is pancreatic islet cells. The invention also provides methodsfor treating type 1 and/or type 2 diabetes in subjects by inhibitingislet cell transplant rejection.

The present invention further provides a method for inhibitingpancreatic islet transplant rejection in a subject, the subject being arecipient of transplant tissue. Typically, in tissue transplants,rejection of the graft is initiated through its recognition as foreignby T cells, followed by an immune response that destroys the graft.Administration of a soluble CTLA4 molecule in the method of thisinvention inhibits T lymphocyte proliferation and/or cytokine secretion,resulting in reduced tissue destruction and induction ofantigen-specific T cell unresponsiveness that may result in long-termgraft acceptance, without the need for generalized immunosuppression.

A preferred embodiment of the invention comprises use of the solubleCTLA4 mutant molecule L104EA29YIg to regulate functional CTLA4- andCD28-positive cell interactions with B7-positive cells, to treat immunesystem diseases such as diabetes and/or to downregulate immuneresponses. The L104EA29YIg of the invention is a soluble CTLA4 mutantmolecule comprising at least the two amino acid changes, the leucine (L)to glutamic acid (E) at position +104 and the alanine (A) to tyrosine(Y) change at position +29. The L104EA29YIg molecule may encompassfurther mutations beyond the two specified herein.

The method can further comprise administering with the soluble CTLA4mutant molecules, a base immunosuppressive regimen to the subject. Thebase immunosuppressive regimen can include (but is not limited to):cyclosporin, azathioprine, methotrexate, cyclophosphamide, lymphocyteimmune globulin, anti-CD3 antibodies, Rho (D) immune globulin,adrenocorticosteroids, sulfasalzine, FK-506. methoxsalen, mycophenolatemofetil (CELLCEPT), horse anti-human thymocyte globulin (ATGAM),humanized anti-TAC(HAT), basiliximab (SIMULECT), rabbit anti-humanthymocyte globulin (THYMOGLOBULIN), sirolimus, thalidomide,methotrexate, chloroquine, hydroxychloroquine, sulfasalazine,sulphasalazopyrine, leflunomide, gold salts, D-penicillamine,azathioprine, anakinra, infliximab, etanercept, TNFα blockers or abiological agent that targets an inflammatory cytokine. In a preferredembodiment, base immunosuppressive regimen is steroid free. Morepreferably, the base immunosuppressive regimen comprises rapamycin andanti-human IL-2 R mAb.

An embodiment of the invention comprises use of a molecule to block theinteraction between B7 and CTLA4 in conjunction with animmunosuppressive agent to regulate an immune response in order to treatan immune system disease such as diabetes. The molecule used to blockthe B7/CTLA4 interaction may be a soluble CTLA4 such as CTLA4Ig,CTLA4Ig/CD28Ig or L104EA29YIg, a soluble CD28 such as CD28Ig, a solubleB7 (B7-1 or B7-2) such as B7Ig, anti-CTLA4 monoclonal antibodies,anti-CD28 monoclonal antibodies or anti-B7 monoclonal antibodies.

The subjects treated by the present invention include mammaliansubjects, including, human, monkey, ape, dog, cat, cow, horse, goat,pig, rabbit, mouse and rat.

The present invention provides various methods, local or systemic, foradministering the therapeutic compositions of the invention such assoluble CTLA4 molecule alone or in conjunction with an immunosuppressiveagent and/or other therapeutic drug. The methods include intravenous,intramuscular, intraperitoneal, oral, inhalation and subcutaneousmethods, as well as implantable pump, continuous infusion, gene therapy,liposomes, suppositories, topical contact, vesicles, capsules andinjection methods. The therapeutic agent, compounded with a carrier, iscommonly lyophilized for storage and is reconstituted with water or abuffered solution with a neutral pH (about pH 7-8, e.g., pH 7.5) priorto administration.

As is standard practice in the art, the compositions of the inventionmay be administered to the subject in any pharmaceutically acceptableform.

In accordance with the practice of the invention, the methods compriseadministering to a subject the soluble CTLA4 molecules of the inventionto regulate CD28- and/or CTLA4-positive cell interactions withB7-positive cells. The B7-positive cells are contacted with an effectiveamount of the soluble CTLA4 molecules of the invention, or fragments orderivatives thereof, so as to form soluble CTLA4/B7 complexes. Thecomplexes interfere with interaction between endogenous CTLA4 and CD28molecules with B7 family molecules.

The soluble CTLA4 molecules may be administered to a subject in anamount and for a time (e.g., length of time and/or multiple times)sufficient to block endogenous B7 molecules from binding theirrespective ligands, in the subject. Blockage of endogenous B7/ligandbinding thereby inhibiting interactions between B7-positive cells withCD28- and/or CTLA4-positive cells.

Dosage of a therapeutic agent is dependant upon many factors including,but not limited to, the type of tissue affected, the type of autoimmunedisease being treated, the severity of the disease, a subject's healthand response to the treatment with the agents. Accordingly, dosages ofthe agents can vary depending on each subject and the mode ofadministration. The soluble CTLA4 molecules may be administered in anamount from about 0.1 to 100 mg/kg weight of the patient/day.

The invention also encompasses the use of the compositions of theinvention together with other pharmaceutical agents to treat immunesystem diseases. For example, diabetes may be treated with molecules ofthe invention in conjunction with, but not limited to, immunosuppressiveagents such as corticosteroids, cyclosporin (Mathiesen 1989 Cancer Lett.44(2):151-156), prednisone, azathioprine, (R. Handschumacher, in: “DrugsUsed for Immunosuppression” pages 1264-1276), TNFα blockers orantagonists (New England Journal of Medicine, vol. 340: 253-259, 1999;The Lancet vol. 354: 1932-39, 1999, Annals of Internal Medicine, vol.130: 478-486), or any other biological agent targeting any inflammatorycytokine, nonsteroidal antiinflammatory drugs/Cox-2 inhibitors,hydroxychloroquine, sulphasalazopryine, gold salts, etanercept,infliximab, rapamycin, mycophenolate mofetil, azathioprine, tacrolismus,basiliximab, cytoxan, interferon beta-1a, interferon beta-1b, glatirameracetate, mitoxantrone hydrochloride, anakinra and/or other biologics.

The soluble CTLA4 molecules (preferably, L104EA29YIg) can also be usedin combination with one or more of the following agents to regulate animmune response: soluble gp39 (also known as CD40 ligand (CD40L), CD154,T-BAM, TRAP), soluble CD29, soluble CD40; soluble CD80 (e.g. ATCC68627), soluble CD86, soluble CD28 (e.g. 68628), soluble CD56, solubleThy-1, soluble CD3, soluble TCR, soluble VLA-4, soluble VCAM-1, solubleLECAM-1, soluble ELAM-1, soluble CD44, antibodies reactive with gp39(e.g. ATCC-HB-10916, ATCC HB-12055 and ATCC HB-12056), antibodiesreactive with CD40 (e.g. ATCC HB-9110), antibodies reactive with B7(e.g. ATCC HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-3.01, ATCCHB-11341, etc), antibodies reactive with CD28 (e.g. ATCC HB-11944 or mAb9.3 as described by Martin et al (J. Clin. Immun. 4(1):18-22, 1980),antibodies reactive with LFA-1 (e.g. ATCC HB-9579 and ATCC TIB-213),antibodies reactive with LFA-2, antibodies reactive with IL-2,antibodies reactive with IL-12, antibodies reactive with IFN-gamma,antibodies reactive with CD2, antibodies reactive with CD48, antibodiesreactive with any ICAM (e.g., ICAM-1 (ATCC CRL-2252), ICAM-2 andICAM-3), antibodies reactive with CTLA4 (e.g. ATCC HB-304), antibodiesreactive with Thy-1, antibodies reactive with CD56, antibodies reactivewith CD3, antibodies reactive with CD29, antibodies reactive with TCR,antibodies reactive with VLA-4, antibodies reactive with VCAM-1,antibodies reactive with LECAM-1, antibodies reactive with ELAM-1,antibodies reactive with CD44. In certain embodiments, monoclonalantibodies are preferred. In other embodiments, antibody fragments arepreferred. As persons skilled in the art will readily understand, thecombination can include the soluble CTLA4 molecules of the invention andone other immunosuppressive agent, the soluble CTLA4 molecules with twoother immunosuppressive agents, the soluble CTLA4 molecules with threeother immunosuppressive agents, etc. The determination of the optimalcombination and dosages can be determined and optimized using methodswell known in the art.

Some specific combinations include the following: L104EA29YIg and CD80monoclonal antibodies (mAbs); L104EA29YIg and CD86 mAbs; L104EA29YIg,CD80 mAbs, and CD86 mAbs; L104EA29YIg and gp39 mAbs; L104EA29YIg andCD40 mAbs; L104EA29YIg and CD28 mAbs; L104EA29YIg, CD80 and CD86 mAbs,and gp39 mAbs; L104EA29YIg, CD80 and CD86 mAbs and CD40 mAbs; andL104EA29YIg, anti-LFA1 mAb, and anti-gp39 mAb. A specific example of agp39 mAb is MR1. Other combinations will be readily appreciated andunderstood by persons skilled in the art.

The soluble CTLA4 molecules of the invention, for example L104EA29YIg,may be administered as the sole active ingredient or together with otherdrugs in immunomodulating regimens or other anti-inflammatory agentse.g., for the treatment or prevention of allo- or xenograft acute orchronic rejection or inflammatory or autoimmune disorders, or to inducetolerance. For example, it may be used in combination with a calcineurininhibitor, e.g. cyclosporin A or FK506; an immunosuppressive macrolide,e.g. rapamycine or a derivative thereof (e.g.40-O-(2-hydroxy)ethyl-rapamycin); a lymphocyte homing agent, e.g. FTY720or an analog thereof; corticosteroids; cyclophosphamide; azathioprene;methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolicacid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof;immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies toleukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD 11a/CD18, CD7, CD25,CD 27, B7, CD40, CD45, CD58, CD 137, ICOS, CD150 (SLAM), OX40, 4-1BB ortheir ligands; or other immunomodulatory compounds, e.g. CTLA4/CD28-Ig,or other adhesion molecule inhibitors, e.g. mAbs or low molecular weightinhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4antagonists. The compound is particularly useful in combination with acompound that interferes with CD40 and its ligand, e.g. antibodies toCD40 and antibodies to CD40-L.

Where the soluble CTLA4 mutant molecules of the invention areadministered in conjunction with otherimmunosuppressive/immunomodulatory or anti-inflammatory therapy, e.g. ashereinabove specified, dosages of the co-administered immunosuppressive,immunomodulatory or anti-inflammatory compound will of course varydepending on the type of co-drug employed, e.g. whether it is a steroidor a cyclosporin, on the specific drug employed, on the condition beingtreated and so forth.

In accordance with the foregoing the present invention provides in a yetfurther aspect methods as defined above comprising co-administration,e.g. concomitantly or in sequence, of a therapeutically effective amountof soluble CTLA4 molecules of the invention, e.g. CTLA4Ig and/orL104EA29YIg, in free form or in pharmaceutically acceptable salt form,and a second drug substance, said second drug substance being animmunosuppressive, immunomodulatory or anti-inflammatory drug, e.g. asindicated above.

Further provided are therapeutic combinations, e.g. a kit, comprising asoluble CTLA4 molecule, in free form or in pharmaceutically acceptablesalt form, to be used concomitantly or in sequence with at least onepharmaceutical composition comprising an immunosuppressive,immunomodulatory or anti-inflammatory drug e.g., NSAID, glucocorticoidor corticosteroid. The kit may comprise instructions for itsadministration. The kits of the invention can be used in any method ofthe present invention.

In another embodiment of the invention, rejection of tissue or organtransplant is inhibited by administering to a subject soluble CTLA4 andT cell depleted bone marrow cells to the subject. Administration of Tcell depleted bone marrow can occur at approximately the same time thatthe subject receives the tissue or organ transplant or at a differenttime. Administration of bone marrow at approximately the same timeindicates that the bone marrow is administered to the subject as part ofthe preparation for the procedures for administering the tissue or organtransplant. It is not required that the bone marrow be transplanted atexactly the same time (i.e., within minutes of) as the organ transplant.

In preferred embodiments, the T cell depleted bone marrow isadministered before the organ transplant. Particular embodiments includeadministering the T cell depleted bone marrow within a day, withintwelve hours, or within six hours of the solid organ transplant.However, the T cell depleted bone marrow can be administered earlier, solong as the resulting effects of the T cell depleted bone marrow arestill achieved in connection with the organ or tissue transplant. Inalternative embodiments, it may be desired to administer T cell depletedbone marrow after the organ transplant.

In one embodiment, the method comprises administering a dose of T celldepleted bone marrow cells (tolerizing dose) to a subject, andsubsequently administering an additional dose of T cell depleted bonemarrow cells (engrafting dose) to the subject. In certain embodiments,the immunosuppressive agent comprises at least one or more types ofligands that interfere with the binding of CD28 antigen to CD80 and/orCD86 antigen. As described supra, the ligand is preferably a mutantCTLA4 molecule, such as L104EA29YIg.

Furthermore, the amount of T cell depleted bone marrow may be determinedby routine experimentation and optimized empirically. For example, theamount of T cell depleted bone marrow can be titrated during routineexperimentation to determine the amount sufficient to achieve thedesired effects.

The methods of the invention may also be practiced by administering, inaddition to the soluble CTLA4 mutant molecule, two or more doses of Tcell depleted bone marrow to the subject, alone, or in combination with,one or more immunosuppressive agents.

As discussed herein, in the methods of the invention, administration ofa soluble CTLA4 or mutant CTLA4 molecule can be accomplished in manydifferent ways including local or systemic administration routes. Forexample, soluble CTLA4 mutant molecules can be administeredintravenously, intramuscularly, or intraperitoneally. Alternatively,mutant CTLA4 may be administered orally or subcutaneously. Other methodsof administration will be recognized by those skilled in the art.Similarly, T cell depleted bone marrow can administered in manydifferent ways as known by persons skilled in the art. One example is byway of intravenous infusion.

The immunosuppressive agent(s) can be administered before or afteradministration of soluble CTLA4 mutant and/or before or after theorgan/tissue transplant. Preferably, the bone marrow andimmunosuppressive agent are administered before administration ofsoluble CTLA4 mutant molecule. In one embodiment, a first dose of T celldepleted bone marrow (tolerizing dose) and the immunosuppressive agentis administered at approximately the same time as the organ transplant.

The following examples are presented to illustrate the presentinvention. The methodology and results may vary depending on theintended goal of treatment and the procedures employed. The examples arenot intended in any way to otherwise limit the scope of the invention.

EXAMPLES Example 1

This example provides a description of the methods used to generate thenucleotide sequences encoding the soluble CTLA4 mutant molecules of theinvention. A single-site mutant L104EIg was generated and tested forbinding kinetics to CD80 and/or CD86. The L104EIg nucleotide sequencewas used as a template to generate the double-site mutant CTLA4sequence, L104EA29YIg, which was tested for binding kinetics to CD80and/or CD86.

CTLA4Ig Codon Based Mutagenesis:

A mutagenesis and screening strategy was developed to identify mutantCTLA4Ig molecules that had slower rates of dissociation (“off” rates)from binding CD86 molecules. Single-site mutant nucleotide sequenceswere generated using CTLA4Ig (U.S. Pat. Nos. 5,844,095; 5,851,795; and5,885,796; ATCC Accession No. 68629) as a template. Mutagenicoligonucleotide PCR primers were designed for random mutagenesis of aspecific cDNA codon by allowing any base at positions 1 and 2 of thecodon, but only guanine or thymine at position 3 (XXG/T; also known asNNG/T). In this manner, a specific codon encoding an amino acid could berandomly mutated to code for each of the 20 amino acids. In that regard,XXG/T mutagenesis yields 32 potential codons encoding each of the 20amino acids. PCR products encoding mutations in close proximity to−M97-G107 of CTLA4Ig (see FIG. 1 or 2), were digested with SacI/XbaI andsubcloned into similarly cut CTLA4Ig πLN expression vector. This methodwas used to generate the single-site CTLA4 mutant molecule L104EIg.

For mutagenesis in proximity to S25-R33 of CTLA4Ig, a silent NheIrestriction site was first introduced 5′ to this loop, by PCRprimer-directed mutagenesis. PCR products were digested with NheI/XbaIand subcloned into similarly cut CTLA4Ig or L104EIg expression vectors.This method was used to generate the double-site CTLA4 mutant moleculeL104EA29Ig (FIG. 3). In particular, the nucleic acid molecule encodingthe single-site CTLA4 mutant molecule, L104EIg, was used as a templateto generate the double-site CTLA4 mutant molecule, L104EA29YIg.

Example 2

The following provides a description of the screening methods used toidentify the single- and double-site mutant CTLA4 polypeptides,expressed from the constructs described in Example 1, that exhibited ahigher binding avidity for CD80 and CD86 antigens, compared tonon-mutated CTLA4Ig molecules.

Current in vitro and in vivo studies indicate that CTLA4Ig by itself isunable to completely block the priming of antigen specific activated Tcells. In vitro studies with CTLA4Ig and either monoclonal antibodyspecific for CD80 or CD86 measuring inhibition of T cell proliferationindicate that anti-CD80 monoclonal antibody did not augment CTLA4Iginhibition. However, anti-CD86 monoclonal antibody did augment theinhibition, indicating that CTLA4Ig was not as effective at blockingCD86 interactions. These data support earlier findings by Linsley et al.(Immunity, (1994), 1:793-801) showing inhibition of CD80-mediatedcellular responses required approximately 100 fold lower CTLA4Igconcentrations than for CD86-mediated responses. Based on thesefindings, it was surmised that soluble CTLA4 mutant molecules having ahigher avidity for CD86 than wild type CTLA4 should be better able toblock the priming of antigen specific activated cells than CTLA4Ig.

To this end, the soluble CTLA4 mutant molecules described in Example 1above were screened using a novel screening procedure to identifyseveral mutations in the extracellular domain of CTLA4 that improvebinding avidity for CD80 and CD86. This screening strategy provided aneffective method to directly identify mutants with apparently slower“off” rates without the need for protein purification or quantitationsince “off” rate determination is concentration independent (O'Shannessyet al., (1993) Anal. Biochem., 212:457-468).

COS cells were transfected with individual miniprep purified plasmid DNAand propagated for several days. Three day conditioned culture media wasapplied to BIAcore biosensor chips (Pharmacia Biotech AB, Uppsala,Sweden) coated with soluble CD80Ig or CD86Ig. The specific binding anddissociation of mutant proteins was measured by surface plasmonresonance (O'Shannessy, D. J., et al., (1993) Anal. Biochem.212:457-468). All experiments were run on BIAcore™ or BIAcore™ 2000biosensors at 25° C. Ligands were immobilized on research grade NCM5sensor chips (Pharmacia) using standard N-ethyl-N′-(dimethylaminopropyl)carbodiimidN-hydroxysuccinimide coupling (Johnsson, B., et al. (1991)Anal. Biochem. 198: 268-277; Khilko, S, N., et al. (1993) J. Biol. Chem.268:5425-15434).

Screening Method

COS cells grown in 24 well tissue culture plates were transientlytransfected with DNA encoding mutant CTLA4 μg. Culture media containingsecreted soluble mutant CTLA4Ig was collected 3 days later.

Conditioned. COS cell culture media was allowed to flow over BIAcorebiosensor chips derivatized with CD86Ig or CD80Ig (as described inGreene et al., 1996 J. Biol. Chem. 271:26762-26771), and mutantmolecules were identified with “off” rates slower than that observed forwild type CTLA4Ig. The cDNAs corresponding to selected media sampleswere sequenced and DNA was prepared to perform larger scale COS celltransient transfection, from which mutant CTLA4Ig protein was preparedfollowing protein A purification of culture media.

BIAcore analysis conditions and equilibrium binding data analysis wereperformed as described in J. Greene et al. 1996 J. Biol. Chem.271:26762-26771, and as described herein.

BIAcore Data Analysis

Senosorgram baselines were normalized to zero response units (RU) priorto analysis. Samples were run over mock-derivatized flow cells todetermine background response unit (RU) values due to bulk refractiveindex differences between solutions. Equilibrium dissociation constants(K_(d)) were calculated from plots of R_(eq) versus C, where R_(eq) isthe steady-state response minus the response on a mock-derivatized chip,and C is the molar concentration of analyte. Binding curves wereanalyzed using commercial nonlinear curve-fitting software (Prism,GraphPAD Software).

Experimental data were first fit to a model for a single ligand bindingto a single receptor (1-site model, i.e., a simple langmuir system, A+BAB), and equilibrium association constants (K_(d)=[A]·[B]\[AB]) werecalculated from the equation R=R_(max)·C/(K_(d)+C). Subsequently, datawere fit to the simplest two-site model of ligand binding (i.e., to areceptor having two non-interacting independent binding sites asdescribed by the equationR=R_(max1)·C\(K_(d1)+C)+R_(max2)·C\(K_(d2)+C)).

The goodness-of-fits of these two models were analyzed visually bycomparison with experimental data and statistically by an F test of thesums-of-squares. The simpler one-site model was chosen as the best fit,unless the two-site model fit significantly better (p<0.1).

Association and disassociation analyses were performed using BIAevaluation 2.1 Software (Pharmacia). Association rate constants k_(on)were calculated in two ways, assuming both homogenous single-siteinteractions and parallel two-site interactions. For single-siteinteractions, k_(on) values were calculated according to the equationR_(t)=R_(eq)(1−exp^(−ks(t-t) ₀), where R_(t) is a response at a giventime, t; R_(eq) is the steady-state response; t₀ is the time at thestart of the injection; and k_(s)=dR/dt=k_(on)·Ck_(off), and where C isa concentration of analyte, calculated in terms of monomeric bindingsites. For two-site interactions k_(on) values were calculated accordingto the equation R_(t)=R_(eq1)(1−exp^(−ks1(t-t)₀)+R_(eq2)(1−exp^(ks2(t-t) ₀). For each model, the values of k_(on) weredetermined from the calculated slope (to about 70% maximal association)of plots of k_(s) versus C.

Dissociation data were analyzed according to one site (AB=A+B) or twosites (AiBj=Ai+Bj) models, and rate constants (k_(off)) were calculatedfrom best fit curves. The binding site model was used except when theresiduals were greater than machine background (2-10 RU, according tomachine), in which case the two-binding site model was employed.Half-times of receptor occupancy were calculated using the relationshipt_(1/2)=0.693/k_(off).

Flow Cytometry:

Murine mAb L307.4 (anti-CD80) was purchased from Becton Dickinson (SanJose, Calif.) and IT2.2 (anti-B7-0 [also known as CD86]), fromPharmingen (San Diego, Calif.). For immunostaining, CD80-positive and/orCD86-positive CHO cells were removed from their culture vessels byincubation in phosphate-buffered saline (PBS) containing 10 mM EDTA. CHOcells (1−10×10⁵) were first incubated with mAbs or immunoglobulin fusionproteins in DMEM containing 10% fetal bovine serum (FBS), then washedand incubated with fluorescein isothiocyanate-conjugated goat anti-mouseor anti-human immunoglobulin second step reagents (Tago, Burlingame,Calif.). Cells were given a final wash and analyzed on a FACScan (BectonDickinson).

SDS-PAGE and Size Exclusion Chromatography

SDS-PAGE was performed on Tris/glycine 4-20% acrylamide gels (Novex, SanDiego, Calif.). Analytical gels were stained with Coomassie Blue, andimages of wet gels were obtained by digital scanning. CTLA4Ig (25 μg)and L104EA29YIg (25 μg) were analyzed by size exclusion chromatographyusing a TSK-GEL G300 SW_(XL) Column (7.8×300 mm, Tosohaas,Montgomeryville, Pa.) equilibrated in phosphate buffered salinecontaining 0.02% NAN₃ at a flow rate of 1.0 ml/min.

CTLA4X_(C120S) and L104EA29YX_(C120S).

Single chain CTLA4X_(C120S) was prepared as previously described(Linsley et al., (1995) J. Biol. Chem., 270:15417-15424). Briefly, anoncostatin M CTLA4 (OMCTLA4) expression plasmid was used as a template,the forward primer, GAGGTGATAAAGCTTCACCAATGGGTGTACTGCTCACACAG (SEQ IDNO.: 17) was chosen to match sequences in the vector; and the reverseprimer, GTGGTGTATTGGTCTAGATCAATCAGAATCTGGGCACGGTTC (SEQ ID NO.: 18)corresponded to the last seven amino acids (i.e. amino acids 118-124) inthe extracellular domain of CTLA4, and contained a restriction enzymesite, and a stop codon (TGA). The reverse primer specified a C120S(cysteine to serine at position 120) mutation. In particular, thenucleotide sequence GCA (nucleotides 34-36) of the reverse primer shownabove is replaced with one of the following nucleotide sequences: AGA,GGA, TGA, CGA, ACT, or GCT. As persons skilled in the art willunderstand, the nucleotide sequence GCA is a reversed complementarysequence of the codon TGC for cysteine. Similarly, the nucleotidesequences AGA, GGA, TGA, CGA, ACT, or GCT are the reversed complementarysequences of the codons for serine. Polymerase chain reaction productswere digested with HindIII/XbaI and directionally subcloned into theexpression vector πLN (Bristol-Myers Squibb Company, Princeton, N.J.).L104EA29YX_(C120S) was prepared in an identical manner. Each constructwas verified by DNA sequencing.

Identification and Biochemical Characterization of High Avidity Mutants

Twenty four amino acids were chosen for mutagenesis and the resulting˜2300 mutant proteins assayed for CD86Ig binding by surface plasmonresonance (SPR; as described, supra). The predominant effects ofmutagenesis at each site are summarized in Table II. Random mutagenesisof some amino acids in the S25-R33 apparently did not alter ligandbinding. Mutagenesis of E31 and R33 and residues M97-Y102 apparentlyresulted in reduced ligand binding. Mutagenesis of residues, S25, A29,and T30, K93, L96, Y103, L104, and G105, resulted in proteins with slow“on” and/or slow “off” rates. These results confirm previous findingsthat residues in the S25-R33 region, and residues in or near M97-Y102influence ligand binding (Peach et al., (1994) J. Exp. Med.180:2049-2058.

Mutagenesis of sites S25, T30, K93, L96, Y103, and G105 resulted in theidentification of some mutant proteins that had slower “off” rates fromCD86Ig. However, in these instances, the slow “off” rate was compromisedby a slow “on” rate which resulted in mutant proteins with an overallavidity for CD86Ig that was apparently similar to that seen with wildtype CTLA4Ig. In addition, mutagenesis of K93 resulted in significantaggregration which may have been responsible for the kinetic changesobserved.

Random mutagenesis of L104 followed by COS cell transfection andscreening by SPR of culture media samples over immobilized CD86Igyielded six media samples containing mutant proteins with approximately2-fold slower “off” rates than wild type CTLA4Ig. When the correspondingcDNA of these mutants were sequenced, each was found to encode a leucineto glutamic acid mutation (L104E). Apparently, substitution of leucine104 to aspartic acid (L104D) did not affect CD86Ig binding.

Mutagenesis was then repeated at each site listed in Table II, this timeusing L104E as the PCR template instead of wild type CTLA4Ig, asdescribed above. SPR analysis, again using immobilized CD86Ig,identified six culture media samples from mutagenesis of alanine 29 withproteins having approximately 4-fold slower “off” rates than wild typeCTLA4Ig. The two slowest were tyrosine substitutions (L104EA29Y), twowere leucine (L104EA29L), one was tryptophan (L104EA29W), and one wasthreonine (L104EA29T). Apparently, no slow “off” rate mutants wereidentified when alanine 29 was randomly mutated, alone, in wild typeCTLA4Ig.

The relative molecular mass and state of aggregration of purified L104Eand L104EA29YIg was assessed by SDS-PAGE and size exclusionchromatography. L104EA29YIg (˜1 μg; lane 3) and L104EIg (˜1 μg; lane 2)apparently had the same electrophoretic mobility as CTLA4Ig (˜1 μg;lane 1) under reducing (˜50 kDa; +βME; plus 2-mercaptoethanol) andnon-reducing (˜100 kDa; −βME) conditions (FIG. 14A). Size exclusionchromatography demonstrated that L104EA29YIg (FIG. 14C) apparently hadthe same mobility as dimeric CTLA4Ig (FIG. 14B). The major peaksrepresent protein dimer while the faster eluting minor peak in FIG. 14Brepresents higher molecular weight aggregates. Approximately 5.0% ofCTLA4Ig was present as higher molecular weight aggregates but there wasno evidence of aggregation of L104EA29YIg or L104EIg. Therefore, thestronger binding to CD86Ig seen with L104EIg and L104EA29YIg could notbe attributed to aggregation induced by mutagenesis.

Equilibrium and Kinetic Binding Analysis

Equilibrium and kinetic binding analysis was performed on protein Apurified CTLA4Ig, L104EIg, and L104EA29YIg using surface plasmonresonance (SPR). The results are shown in Table I. Observed equilibriumdissociation constants (K_(d); Table I) were calculated from bindingcurves generated over a range of concentrations (5.0-200 nM).L104EA29YIg binds more strongly to CD86Ig than does L104EIg or CTLA4Ig.The lower K_(d) of L104EA29YIg (3.21 nM) than L104EIg (6.06 nM) orCTLA4Ig (13.9 nM) indicates higher binding avidity of L104EA29YIg toCD86Ig. The lower K_(d) of L104EA29YIg (3.66 nM) than L104EIg (4.47 nM)or CTLA4Ig (6.51 nM) indicates higher binding avidity of L104EA29YIg toCD80Ig.

Kinetic binding analysis revealed that the comparative “on” rates forCTLA4Ig, L104EIg, and L104EA29YIg binding to CD80 were similar, as werethe “on” rates for CD86Ig (Table I). However, “off” rates for thesemolecules were not equivalent (Table I). Compared to CTLA4Ig,L104EA29YIg had approximately 2-fold slower “off” rate from CD80Ig, andapproximately 4-fold slower “off” rate from CD86Ig. L104E had “off”rates intermediate between L104EA29YIg and CTLA4Ig. Since theintroduction of these mutations did not significantly affect “on” rates,the increase in avidity for CD80Ig and CD86Ig observed with L104EA29YIgwas likely primarily due to a decrease in “off” rates.

To determine whether the increase in avidity of L104EA29YIg for CD86Igand CD80Ig was due to the mutations affecting the way each monomerassociated as a dimer, or whether there were avidity enhancingstructural changes introduced into each monomer, single chain constructsof CTLA4 and L104EA29Y extracellular domains were prepared followingmutagenesis of cysteine 120 to serine as described supra, and by Linsleyet al., (1995) J. Biol. Chem., 270:15417-15424. The purified proteinsCTLA4X_(C120S) and L104EA29YX_(C120S) were shown to be monomeric by gelpermeation chromatography (Linsley et al., (1995), supra), before theirligand binding properties were analyzed by SPR. Results showed thatbinding affinity of both monomeric proteins for CD86Ig was approximately35-80-fold less than that seen for their respective dimers (Table I).This supports previously published data establishing that dimerizationof CTLA4 was required for high avidity ligand binding (Greene et al.,(1996) J. Biol. Chem., 271:26762-26771). L104EA29YX_(C120S) bound withapproximately 2-fold higher affinity than CTLA4X_(C120S) to both CD80Igand CD86Ig. The increased affinity was due to approximately 3-foldslower rate of dissociation from both ligands. Therefore, strongerligand binding by L104EA29Y was most likely due to avidity enhancingstructural changes that had been introduced into each monomeric chainrather than alterations in which the molecule dimerized.

Location and Structural Analysis of Avidity Enhancing Mutations

The solution structure of the extracellular IgV-like domain of CTLA4 hasrecently been determined by NMR spectroscopy (Metzler et al., (1997)Nature Struct. Biol., 4:527-531. This allowed accurate location ofleucine 104 and alanine 29 in the three dimensional fold (FIG. 15A-B).Leucine 104 is situated near the highly conserved MYPPPY amino acidsequence. Alanine 29 is situated near the C-terminal end of the S25-R33region, which is spatially adjacent to the MYPPPY region. While there issignificant interaction between residues at the base of these tworegions, there is apparently no direct interaction between L104 and A29although they both comprise part of a contiguous hydrophobic core in theprotein. The structural consequences of the two avidity enhancingmutants were assessed by modeling. The A29Y mutation can be easilyaccommodated in the cleft between the S25-R33 region and the MYPPPYregion, and may serve to stabilize the conformation of the MYPPPYregion. In wild type CTLA4, L104 forms extensive hydrophobicinteractions with L96 and V94 near the MYPPPY region. It is highlyunlikely that the glutamic acid mutation adopts a conformation similarto that of L104 for two reasons. First, there is insufficient space toaccommodate the longer glutamic acid side chain in the structure withoutsignificant perturbation to the S25-R33 region. Second, the energeticcosts of burying the negative charge of the glutamic acid side chain inthe hydrophobic region would be large. Instead, modeling studies predictthat the glutamic acid side chain flips out on to the surface where itscharge can be stabilized by solvation. Such a conformational change caneasily be accommodated by G105, with minimal distortion to otherresidues in the regions.

Binding of High Avidity Mutants to CHO Cells Expressing CD80 or CD86

FACS analysis (FIG. 9) of CTLA4Ig and mutant molecules binding to stablytransfected CD80+ and CD86+CHO cells was performed as described herein.CD80-positive and CD86-positive CHO cells were incubated with increasingconcentrations of CTLA4Ig, L104EA29YIg, or L104EIg, and then washed.Bound immunoglobulin fusion protein was detected using fluoresceinisothiocyanate-conjugated goat anti-human immunoglobulin.

As shown in FIG. 9, CD80-positive or CD86-positive CHO cells (1.5×10⁵)were incubated with the indicated concentrations of CTLA4Ig (closedsquares), L104EA29YIg (circles), or L104EIg (triangles) for 2 hr. at 93°C., washed, and incubated with fluorescein isothiocyanate-conjugatedgoat anti-human immunoglobulin antibody. Binding on a total of 5,000viable cells was analyzed (single determination) on a FACScan, and meanfluorescence intensity (MFI) was determined from data histograms usingPC-LYSYS. Data were corrected for background fluorescence measured oncells incubated with second step reagent only (MFI=7). Control L6 mAb(80 μg/ml) gave MFI<30. These results are representative of fourindependent experiments.

Binding of L104EA29YIg, L104EIg, and CTLA4Ig to human CD80-transfectedCHO cells is approximately equivalent (FIG. 9A). L104EA29YIg and L104EIgbind more strongly to CHO cells stably transfected with human CD86 thandoes CTLA4Ig (FIG. 9B).

Functional Assays:

Human CD4-positive T cells were isolated by immunomagnetic negativeselection (Linsley et al., (1992) J. Exp. Med. 176:1595-1604). IsolatedCD4-positive T cells were stimulated with phorbal myristate acetate(PMA) plus CD80-positive or CD86-positive CHO cells in the presence oftitrating concentrations of inhibitor. CD4-positive T cells(8−10×10⁴/well) were cultured in the presence of 1 nM PMA with orwithout irradiated CHO cell stimulators. Proliferative responses weremeasured by the addition of 1 μCi/well of [3H]thymidine during the final7 hours of a 72 hour culture. Inhibition of PMA plus CD80-positive CHO,or CD86-positive CHO, stimulated T cells by L104EA29YIg and CTLA4Ig wasperformed. The results are shown in FIG. 10. L104EA29YIg inhibitsproliferation of CD80-positive PMA treated CHO cells more than CTLA4Ig(FIG. 10A). L104EA29YIg is also more effective than CTLA4Ig atinhibiting proliferation of CD86-positive PMA treated CHO cells (FIG.10B). Therefore, L104EA29YIg is a more potent inhibitor of both CD80-and CD86-mediated costimulation of T cells.

FIG. 11 shows inhibition by L104EA29YIg and CTLA4Ig of allostimulatedhuman T cells prepared above, and further allostimulated with a human Blymphoblastoid cell line (LCL) called PM that expressed CD80 and CD86 (Tcells at 3.0×10⁴/well and PM at 8.0×10³/well). Primary allostimulationoccurred for 6 days, then the cells were pulsed with ³H-thymidine for 7hours, before incorporation of radiolabel was determined.

Secondary allostimulation was performed as follows. Seven day primaryallostimulated T cells were harvested over lymphocyte separation medium(LSM) (ICN, Aurora, Ohio) and rested for 24 hours. T cells were thenrestimulated (secondary), in the presence of titrating amounts ofCTLA4Ig or L104EA29YIg, by adding PM in the same ratio as above.Stimulation occurred for 3 days, then the cells were pulsed withradiolabel and harvested as above. The effect of L104EA29YIg on primaryallostimulated T cells is shown in FIG. 11A. The effect of L104EA29YIgon secondary allostimulated T cells is shown in FIG. 11B. L104EA29YIginhibits both primary and secondary T cell proliferative responsesbetter than CTLA4Ig.

To measure cytokine production (FIG. 12), duplicate secondaryallostimulation plates were set up. After 3 days, culture media wasassayed using ELISA kits (Biosource, Camarillo, Calif.) using conditionsrecommended by the manufacturer. L104EA29YIg was found to be more potentthan CTLA4Ig at blocking T cell IL-2, IL-4, and Y-IFN cytokineproduction following a secondary allogeneic stimulus (FIGS. 12A-C).

The effects of L104EA29YIg and CTLA4Ig on monkey mixed lymphocyteresponse (MLR) are shown in FIG. 13. Peripheral blood mononuclear cells(PBMC'S; 3.5×10⁴ cells/well from each monkey) from 2 monkeys werepurified over lymphocyte separation medium (LSM) and mixed with 2 μg/mlphytohemaglutinin (PHA). The cells were stimulated 3 days then pulsedwith radiolabel 16 hours before harvesting. L104EA29YIg inhibited monkeyT cell proliferation better than CTLA4Ig.

TABLE I Equilibrium and apparent kinetic constants are given in thefollowing table (values are means ± standard deviation from threedifferent experiments): k_(on) (×10⁵) K_(d) Immobilized Protein AnalyteM⁻¹ S⁻¹ k_(off) (×10⁻³) S⁻¹ nM CD80Ig CTLA4Ig 3.44 ± 0.29 2.21 ± 0.186.51 ± 1.08 CD80Ig L104EIg 3.02 ± 0.05 1.35 ± 0.08 4.47 ± 0.36 CD80IgL104EA29YIg 2.96 ± 0.20 1.08 ± 0.05 3.66 ± 0.41 CD80Ig CTLA4X_(C120S)12.0 ± 1.0  230 ± 10  195 ± 25  CD80Ig L104EA29YX_(C120S)  8.3 ± 0.26 71± 5  85.0 ± 2.5  CD86Ig CTLA4Ig 5.95 ± 0.57 8.16 ± 0.52 13.9 ± 2.27CD86Ig L104EIg 7.03 ± 0.22 4.26 ± 0.11 6.06 ± 0.05 CD86Ig L104EA29YIg6.42 ± 0.40 2.06 ± 0.03 3.21 ± 0.23 CD86Ig CTLA4X_(C120S) 16.5 ± 0.5 840 ± 55  511 ± 17  CD86Ig L104EA29YX_(C120S) 11.4 ± 1.6  300 ± 10  267± 29 

TABLE II The effect on CD86Ig binding by mutagenesis of CTLA4Ig at thesites listed was determined by SPR, described supra. The predominanteffect is indicated with a “+” sign. Effects of Mutagenesis MutagenesisSlow “on” rate/slow Reduced ligand Site No Apparent Effect “off ratebinding S25 + P26 + G27 + K28 + A29 + T30 + E31 + R33 + K93 + L96 +M97 + Y98 + P99 + P100 + P101 + Y102 + Y103 + L104 + G105 + I106 +G107 + Q111 + Y113 + I115 +

Example 3

This example provides a description of donor pancreatectomy and isletisolation, and islet transplant procedures in an animal model.

Materials and Method:

Animals. Captive bred adolescent male rhesus monkeys (Macaca mulatta)(˜4-20 kg) were used as recipients and donors. The absence of preformeddonor-specific antibodies in the recipient was confirmed prior totransplant. All potential donors and recipients were tested for anti-CMVantibodies and only animals that were sero-positive for CMV were used asrecipients.

Donor Pancreatectomy and Islet Isolation. The donor pancreatectomy wasperformed one day prior to transplantation. The procedure was performedunder general anesthesia (a combination of parenteral ketamine andIsolflurane by inhalation) through a midline abdominal incision. Thesplenorenal and splenocolic ligaments were divided so that the spleen,together with the tail of the pancreas was mobilized. The head of thepancreas and second portion of the duodenum were mobilized followingKocher maneuver. After administration of heparin (200 U/kg), the aortawas cannulated just above its bifurcation and the animal wasexsanguinated. Cold slush was immediately placed in the lesser sac andbehind the body of the pancreas. The body and neck of the pancreas werecarefully excised by sharp dissection taking care not to violate thepancreatic capsule. The common bile duct, the main and accessorypancreatic ducts were identified and ligated, and the head of thepancreas dissected from the second portion of the duodenum.

Rhesus monkey islet isolation was completed via minor modifications ofthe automated method for human islet isolation (Ricordi, (1988)Diabetes, 37: 413; Ranuncoli, (2000) Cell Transplant 9: 409) by usingLiberase (Roche/Boehringer Mannheim, Indpls, Ind.) at a concentration of0.47-0.71 mg/ml. A three layer, discontinuous Euroficoll gradient(densities 1.108, 1.097, 1.037; Meditech, Herndon, Va.) and a Cobe 2991blood cell processor (Gambro, Lakewood, Colo.) were used forpurification of islets from the pancreatic digest. Samples of the finalislet preparation were stained with dithizone (Sigma, St. Louis, Mo.),and the preparation was assessed by counting the number of islets ineach of the following size ranges: 50-100, 100-150, 150-200, 200-250,250-300, 300-350, and 350-400 μm. The data were mathematically convertedto determine the number of islets with an average diameter of 150 μm andwere expressed as islet equivalents (IEQ) (Ricordi C. et al., ActaDiabetol Lat 27:185-195, 1990).

Recipient Pancreatectomy and Intrahepatic Islet Cell Transplantation.Total pancreatectomy, without duodenectomy or splenectomy, was performedat least one week prior to transplant. The tail and body of the pancreaswere dissected along the splenic artery and vein, which were carefullypreserved by ligating and dividing only pancreatic branches. Theinferior mesenteric and middle colic veins were identified and preservedduring dissection of the body of the pancreas. The portal and superiormesenteric veins were recognized and pancreatic veins were ligated anddivided.

The duodenum was mobilized and branches of the pancreaticoduodenalvessels that entered the pancreas were ligated and divided, leaving theduodenal branches intact. The common bile duct was identified andpreserved during blunt dissection between the head of the pancreas andthe c-loop of the duodenum. The main and accessory pancreatic ducts wereligated, divided, and the pancreas was removed from the abdominalcavity. All animals underwent intravenous glucose tolerance test toevaluate the efficacy of the pancreatectomy procedure. All weredocumented to be c-peptide negative prior to islet transplantation.

Overnight cultured islets were washed in transplant media, consisting ofRPMI medium 1640 (Mediatech) supplemented with 2.5% human serum albumin,and counted to determine the number of IEQ. Islets were then pelletedand resuspended in 20 ml of transplant media supplemented with 200 unitsof heparin. Intra-hepatic islet transplantation was performed viagravity drainage of islets into a sigmoid or branch of the left colicvein draining into the portal vein through a 22-gauge intravenouscatheter.

Blood glucose monitoring, insulin administration, and definition ofrejection. Fasting and post-prandial blood glucose levels were monitoredtwice daily (pre-breakfast and post-lunch) via ear-stick, followed byblood testing with a glucometer elite (Bayer, Elkhart, Ind.). Insulin(NPH, Ultralente; Eli Lilly, Indianpolis, Ind.) was administered threetimes daily in attempt to maintain fasting blood glucose<300 mg/dl inpretransplant pancreatectomized animals or in those who had rejectedtheir allografts.

Experimental groups and immunosuppressive protocols. Two treatmentprotocols were tested: (1) Edmonton protocol—using Tacrolimus,Sirolimus, and anti-IL-2R in Ab (Shapiro, A. M. J. et al, (2000), N.Eng. J. Med., 343: 230-238) and (2) L104EA29Y-Edmonton protocol—usingL104EA29YIg, Sirolimus, and anti-IL-2R mAb. The control group includedrecipients treated with “base immunosuppressive regimen” havingrapamycin and anti-IL-2R alone. Tacrolimus was given 0.05 mg/kg bid POD0-14 (target levels 5-8) and 0.06 mg/kg daily (target levels 3-5) POD15-120. L104EA29YIg was administered intravenously intra-operatively (10mg/kg) and on post-operative days 4 (15 mg/kg), 14, 28, 42, 56, 70, 84,98, 112, 126 (20 mg/kg) to maintain serum trough levels greater than 30μg/ml. The chimeric anti-human IL-2R mAb (0.3 mg/kg iv), wasadministered intra-operatively and on POD 4. Sirolimus (Rapamune®) wasadministered orally 1.25 mg/kg bid (target levels 10-15) POD 0-50, 1mg/kg bid (target levels 7-10) POD 50-100, and then tapered to terminatedosing by POD130, Sirolimus (Rapamune®) and Tacrolimus (Prograf®) werepurchased from the Emory University Hospital Pharmacy. The chimericanti-human IL-2R mAb (Simulect®) was provided by Novartis Pharma AG(Basel, Switzerland).

Necropsy. All recipients had a complete necropsy performed by the YerkesVeterinary Staff at the time of their death.

Detection of Anti-donor antibodies. The presence of detectable donorspecific alloantibody was determined using flow cytometry. Peripheralblood leukocytes served as the target cells for the pre-transplantanalysis. Leukocytes isolated from mesenteric lymph nodes obtained atthe time of transplant were the target cells for the post-transplantassays.

Statistics. Survival of the islet grafts among experimental groups wascompared using the Mann-Whitney-Wilcoxon test (Armitage et al. (1987)Statistical methods in Medical Research, Blackwell ScientificPublication, Oxford).

Anti-donor enzyme-linked immunospot assay. Responses were measured byinterferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay usingperipheral blood leukocytes obtained from the recipient and donoranimals. An equal number of irradiated stimulators (donor leukocytes)and responders (recipient leukocytes) were added to ester cellulosebottom plates (Millipore, Bedford, Mass.) coated with the captureantibody, mouse anti-human IFN-γ (clone GZ-4; Mabtech, Sweden). After14-16 h incubation, biotinylated mouse anti-human IFN-γ (clone 7-B6-1;Mabtech, Sweden) was added, unbound antibody was removed, andhorseradish peroxidase-Avidin D (Vector, Burlingame, Calif.) was added.Spots were developed with the substrate 3-amino-9-ethyl-carbazole(Sigma). Each spot represents an IFN-γ-secreting cell; the frequency ofthese cells can be determined by dividing the number of spots generatedby the total number of responder cells plated.

Results:

CD28 pathway blockade-based therapy prolongs the survival of the isletallografts in Rhesus macaques. Diabetes was induced by surgicalpancreatectomy of recipient animals and confirmed by pretransplantintravenous glucose tolerance test. Donor-recipient pairings weredefined based on molecular typing using a panel of previously definedmajor histocompatibilty alleles (8 class 1 and 12 class II) (LobashevskyA, et al., Tissue Antigens 54:254-263, (1999); Knapp L A, et al., TissueAntigens 50:657-661, (1997); Watkins D. I., Crit. Rev Immunol 15:1-29,(1995)). Pairings maximized disparity at both class I and II loci.Rejection was defined as two consecutive fasting blood glucosevalues>125 mg/dl on subsequent days. Intra-portal infusion of allogeneicislets (>10,000 IEQ/Kg) resulted in initial restoration of euglycemiaand insulin independence in diabetic monkeys in both groups.

Treatment of pancreatectomized macaques with theL104EA29YIg/Rapamycin/anti-IL-2R mAb regimen significantly prolongedislet allograft survival (204, 190, 216, >220 and 56 days,respectively). The animals receiving L104EA29YIg/Rapamycin/anti-IL-2Rregimen resulted in adequate glucose control as indicated by fastingplasma glucose levels (FIGS. 5 and 16B). In addition, these animals didnot require insulin replacement therapy for a significantly prolongedperiod of time (FIG. 6). In contrast, those animals receiving the baseregimen alone (Rapamycin/anti-IL-2R mAb) rejected the transplantedislets within one week (FIG. 16C). The control animals showed markedlyelevated levels of fasting plasma glucose (FIG. 5). Further, the controlanimals required insulin replacement therapy within one week of islettransplant (FIG. 6). Four of five animals receiving the L104EA29YIgregimen enjoyed rejection-free survival for the duration of thetreatment period (Table III). Intravenous glucose tolerance test withmeasurement of insulin and glucose levels confirmed islet functionposttransplant (representative animal, FIGS. 7 and 16D).

TABLE III Islet allograft survival and treatment MHC mismatches (n)IEQ/kg Survival* Treatment Class I Class II RKf-7 22,250 204LEA29Y/Rapa/αIL-2R 2 ND RUf-7 17,087 190 LEA29Y/Rapa/αIL-2R ND 3 RRe-720,266 216 LEA29Y/Rapa/αIL-2R 2 6 RWt-6 16,033 56 LEA29Y/Rapa/αIL-2R 2 3RMv-6  8,201 >220 LEA29Y/Rapa/αIL-2R 1 3 RQz-6 12,980 7 Rapa/αIL-2R 2 5RIb-7 10,903 7 Rapa/αIL-2R 1 4 *Insulin independence. ND, none detectedin alleles that were typed.

At 100 days posttransplant, the dosing of rapamycin was decreased andtapered to zero by day 121. Animals continued to remaininsulin-independent while receiving L104EA29YIg monotherapy. At ˜150days posttransplant, the remaining islet recipients received their finaldose of L104EA29YIg, ceasing any additional immunosuppressive therapy.

As expected, ˜1-2 months after discontinuation of therapy, recipientsbecame hyperglycemic and required exogenous insulin therapy.Histological analysis revealed a mononuclear infiltrate, stronglysuggesting rejection as the etiology of the loss of glucose control(FIG. 17). In an intravenous glucose tolerance test, the test animalsreceiving L104EA29YIg/Rapamycin/anti-IL-2R mAb regimen demonstratednormal glucose levels post islet transplant (FIGS. 7 and 16D).

The frequency of anti-donor IFN-γ producing cells was detected byELISpot assay and analyzed using an Immunspot imaging system. Animalsreceiving the base immunosuppressive regimen alone demonstratedsignificantly increased numbers of donor-reactive IFNγ producing T cells(84±4.6 cells), while animals receiving the L104EA29YIg regimen had nodetectable response (2±0.56 cells).

L104EA29YIg therapy inhibits priming of anti-donor T- and B-cellresponses. The frequency of primed alloreactive T-cells can effectivelybe detected by using the ELISpot assay, which can discriminateproduction of IFN-γ at the single-cell level. Peripheral blood samplesfrom islet recipients were analyzed at various time points both pre- andposttransplant for their ability to generate IFN-γ in response to donorantigen. Animals treated with the base regimen alone quickly developed ameasurable anti-donor response that coincided with rejection 1 weekafter transplant. In contrast, the frequency of anti-donor IFN-γproducing cells in animals receiving the L104EA29YIg-containing regimenwas undetectable until therapy was withdrawn (representative animals,FIGS. 18A and B). Thus, the L104EA29YIg regimen effectively blocked thegeneration of anti-donor T-cell responses as measured by the ability toproduce IFN-γ.

Flow cytometry was used to examine the development of anti-donorantibody responses. One animal within the control group generated astrong anti-donor Ab response, whereas the other failed to develop adetectable response, presumably because it was euthanized before theantibody response could be measured (FIG. 18C). In contrast, four offive animals failed to generate an antibody response while receivingL104EA29YIg therapy. This is consistent with previously reported resultsusing CTLA4-Ig in an islet transplant model (Levisetti, M. G. et al., JImmunol 159:5187-5191, 1997) as well as our experience in a renalallograft model where recipients failed to generate anti-donoranti-bodies (Pearson T, ET AL., (Abstract). In Programs and Abstracts ofthe 17th American Society of Transplant Physicians Annual Meeting,Chicago, 10-14 May 1997. Chicago, American Society of TransplantPhysicians). One animal of five recipients underwent a rejection episodewhile receiving the L104EA29YIg therapy and subsequently developed ananti-donor antibody response. As expected, the remaining four animalsreceiving the L104EA29YIg regimen consistently developed anti-donoranti-body responses around the time of rejection (˜200 daysposttransplant, 50 days after the final dose of L104EA29YIg).

Islet transplantation is quickly becoming a viable treatment option forpatients with brittle type 1 diabetes. Recent reports describingsteroid-free immunosuppressive regimens, which result in successfulinsulin independence after islet transplantation, have ushered inrenewed optimism for the practical application of islet transplantation.Whereas the elimination of glucocorticoids from immunosuppressiveregimens represents a major step forward in the effort to treat type 1diabetes, the reliance on calcineurin inhibitor therapy for primaryimmunosuppression may limit the application of this approach.Calcineurin inhibitors, have numerous unwanted side effects, includingnephrotoxicity, diabetes, hypertension, impaired lipid metabolism, andhirusitism (Kahan B. D. et al., N Engl J Med 321:1725-1738, 1989; GroupTUSMFLS: A comparison of tacrolimus (FK 506) and cyclosporine forimmunosuppression in liver transplantation: the U.S. Multicenter FK506Liver Study Group. N Engl J Med 331:1110-1115, 1994; de Mattos A M etal., Am J Kidney Disease 35:333-346, 2000). Even when drug levels arekept low, significant side effects may develop. This is particularlytrue in the diabetic patient population where renal function may alreadybe impaired. Indeed, in the most recent reports from Edmonton, twopatients with mildly elevated pretransplant creatinine levels hadsignificant decreases in renal function while on calcineurin inhibitortherapy and ultimately required withdrawal of this drug (Ryan E. A., etal., Diabetes 50:710-719, 2001). In the same report, two-thirds ofrecipients developed some degree of glucose intolerance, withone-quarter developing frank posttransplant diabetes thought to berelated to the use of tacrolimus. This underscores the appealing andessential nature of a calcineurin inhibitor-free immunosuppressiveregimen, particularly for islet transplantation.

Blockade of T-cell costimulatory pathways is a promising strategy forthe development of nontoxic immunosuppressive and potentiallytolerogenic regimens. This approach targets those T-cells that receive“signal 1” during the period of drug administration. For example,treatment during the peritransplant period is thought to renderallo-specific T-cells impotent upon encounter with the new organ ortissue, whereas other T-cells are left unimpaired (Li Y, et al., Nat Med5:1298-1302, 1999). Blockade of the CD28/B7 pathway has demonstratedremarkable promise in experimental models of autoimmunity andtransplantation, making it a particularly appealing immunosuppressivetarget in islet transplantation, where presumably both auto- andallo-immune obstacles exist. The potential of CD28 blockade in a largeanimal transplant model was described by Levisetti M G, et al., (JImmunol 159:5187-5191, 1997). Treatment with CTLA4-Ig was found tosignificantly, although modestly, prolong islet allograft survival innonhuman primates (Levisetti M G, et al., J Immunol 159:5187-5191,1997). CTLA4-Ig monotherapy does little to prolong renal allograftsurvival (Pearson T. et al., (Abstract). In Programs and Abstracts ofthe 17th American Society of Transplant Physicians Annual Meeting,Chicago, 10-14 May 1997. Chicago, American Society of TransplantPhysicians). Recently, there have been several reports of long-termsurvival of islet allografts in nonhuman primate models. Anti-CD40L mAbtherapy has shown the most impressive results thus far; however, similarto experiments using a renal transplant model, tolerance was notachieved, as withdrawal of therapy eventually resulted in rejection(Kenyon, N. S. et al., Proc. Natl. Acad. Sci. USA 96:8132-8137 (1999);Kirk, A. D., et al., Nat. Med. 5, 686-693 (1999). In another encouragingreport, Thomas et al. (Diabetes 50:1227-1236, (2001)) recently describedthe use of an anti-CD3 immunotoxin and the immune modulatory agent DSG(15 deoxyspergualin) to dramatically prolong islet survival instreptozotocin-induced diabetic primates. Although promising, thesereports used therapeutics whose clinical potential at the present timeis still uncertain.

The in vivo data using L104EA29YIg, a mutant form of CTLA4-Ig, in theRhesus islet allograft model is consistent with in vitro evidenceindicating that this second generation molecule is a more potentinhibitor of T-cell responses than the parent molecule. Given thatCTLA4-Ig has already shown efficacy in a clinical trial of psoriasispatients (Abrams, J. R. et al., J. Clin. Invest. 103:1243-1252 (1999)),there is significant enthusiasm for the trials using L104EA29YIg as theprimary immunosuppresant. It is clearly compatible, if not synergistic,with clinically approved immunosuppressive agents (anti-IL-2R mAb andrapamycin) facilitating the design of clinical trials. Initial humantrials with L104EA29YIg are already underway in patients afflicted withrheumatoid arthritis and those undergoing renal transplant. Although adirect comparison of a tacrolimus-based protocol and the L104EA29YIgregimen was not attempted because of reported intolerable toxicities innonhuman primates (Montgomery, S. P., et al., Am J. Transplant 1 (Suppl.1):438, 2001), our results suggest that L104EA29YIg has the potential tobe at least as effective as tacrolimus as a primary immunosuppressant.

CONCLUSIONS

A novel calcineurin inhibitor/steroid-free immunosuppressive regimenthat provides significant protection from rejection and prolongs thesurvival of islet allografts in nonhuman primates is described. Thebiologic agent L104EA29YIg is a potent immunosuppressant. L104EA29YIgmay replace Tacrolimus in the Edmonton protocol, thereby eliminating theunwanted side effects of the calcineurin inhibitor.

As will be apparent to those skilled in the art to which the inventionpertains, the present invention may be embodied in forms other thanthose specifically disclosed above without departing from the spirit oressential characteristics of the invention. The particular embodimentsof the invention described above, are, therefore, to be considered asillustrative and not restrictive. The scope of the present invention isas set forth in the appended claims rather than being limited to theexamples contained in the foregoing description.

1. A method for inhibiting islet cell transplant rejection in a subject, comprising administering to the subject an effective amount of a CTLA mutant molecule, wherein the subject being transplanted with islet cells before, or after, administration of the CTLA4 mutant molecule.
 2. The method of claim 1, wherein the mutant CTLA4 molecule is L104EA29YIg (FIG. 3), L104EIg (FIG. 19), L104EA29LIg (FIG. 20), L104EA29TIg (FIG. 21) or L104EA29WIg (FIG. 22).
 3. The method of claim 1, wherein the mutant CTLA4 molecule binds to CD80 and/or CD86 molecule on CD80 and/or CD86-positive cells.
 4. A method for treating diabetes by inhibiting islet cell transplant rejection in a subject by the method of claim
 1. 5. The method of claim 1, wherein the islet cells are encapsulated prior to administration to the subject.
 6. The method of claim 1, further comprising administering to the subject an effective amount of at least one immunosuppressive agent prior to, during, or after the transplant.
 7. The method of claim 6, wherein the immunosuppressive agent is steroid-free.
 8. The method of claim 6, wherein the immunosuppressive agent comprises Rapamycin and anti-human IL-2R mAb.
 9. The method of claim 6, wherein the immunosuppressive agent is a corticosteroid, cyclosporin, tarcolimus, prednisone, azathioprine, TOR-inhibitor, methotrexate, TNFα blocker, TNF antagonist, infliximab, a biological agent targeting an inflammatory cytokine, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, or anakinra.
 10. The method of claim 1, wherein the mutant CTLA4 molecule is L104EA29YIg (FIG. 3), which interferes with T-cell/CD80 and/or CD86-positive-cell interactions.
 11. The method of claim 1, wherein administration of the mutant CTLA4 molecule, L104EA29YIg (FIG. 3) is effected locally or systemically.
 12. The method of claim 11, wherein administration is by intravenous injection, intramuscular injection, subcutaneous injection, implantable pump, continuous infusion, gene therapy, lipososomes or oral administration.
 13. The method of claim 1, wherein the subject is a human, non-human primate, rabbit, sheep, rat, dog, cat, pig, or mouse.
 14. The method of claim 13, wherein the non-human primate is a monkey.
 15. The method of claim 1 further comprising administering T cell depleted bone marrow cells to the subject. 